Biology
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Item Restricted Investigating the role of microRNAs on the transgenerational effects of nicotine using Caenorhabditis elegans(2025-05-01) Woollen, Victoria RoseThis project aims to investigate the effects of nicotine, the highly addictive and toxic substance in tobacco-based products, has on offspring (1st and 2nd generations, F1 and F2) using the model organism Caenorhabditis elegans (C. elegans). C. elegans are microscopic nematode worms that are used as a model organism to study epigenetic effects passed down through generations. Due to their transparency, small size, and cellular simplicity, researchers can easily observe the internal structures and processes of C. elegans. To characterize nicotine-dependence, our lab looks at three behaviors: stimulation, adaption, and withdrawal. We hypothesize that maternal nicotine exposure (F0 generation) increases susceptibility to nicotine dependence in F1 and F2 generations. To test this, F0 worms were exposed to a 61.7 µM nicotine solution or a vehicle control (K medium) at the L3 larval stage for 24 hours, then the behaviors were observed in F0, F1, and F2 generations, where the F1 and F2 generations were not exposed to nicotine. Locomotion behaviors were analyzed using WormLab. For nicotine-exposed worms, an increased speed in a nicotine-free environment indicates withdrawal, while a lower locomotion speed in nicotine-containing environment indicates nicotine adaption. To explore miRNA-mediated molecular mechanisms, RNA interference with the miRNA inhibitor anti-cel-miR-1 will knock down miR-1, which regulates nicotinic acetylcholine receptor (nAChR) expression. Behavioral changes in miRNA inhibitor and nicotine co-treated worms will be compared to nicotine-only treated worms and vehicle controls. Statistical analyses of locomotion behaviors revealed transgenerational inheritance of nicotine-dependent behaviors and the important role of miRNA-1 in nicotine-dependent behaviors. With miR-1 inhibition in F0 generation, the nicotine-dependent behavior was cured in F0 and the following F1 and F2 generations. This represents an important finding in the mechanism of nicotine-dependent behaviors and potentially applies to the development of future miRNA-mediated therapeutic strategies.Item Restricted Exploring Patient Perspectives Through Visual Research(2025-05-02) Watson, Calissa BrookeWhen discussing the major conflicts and public health concerns regarding oral health, one of the main subjects is dental caries in youth. Dental caries is the most common chronic disease among children ages 6-10 years old (CDC). Between the years 2015 and 2016, 45.8% of youth aged 2-19 years had caries (CDC). Of those individuals, the prevalence increased with age. Not only do these youth have caries, but a fraction of the caries are left untreated. 13.0% of youth aged 2-19 years had untreated caries in the primary or permanent teeth (CDC). When analyzing data associated with dental caries in youth and the federal poverty level, it is shown that dental caries increase in youth from families living below the federal poverty level. Preventive care is necessary in preventing dental caries and preventative care can start during the mother’s pregnancy. However, in some cases, this is not occurring and is negatively impacting the mother’s oral health as well as the infants. This research relies heavily on the voices of those being impacted (the pregnant women or mothers of infants) and those who treat the women (health care providers). Photovoice as well as interviews is the main form of gaining qualitative data. Photovoice is a visual research methodology that allows participants to document and present their concerns in vulnerable communities. Participants are able to find their voice through the use of photos. The finalized research will be able to thoroughly discuss the barriers between pregnant women and oral health. The main objective of this research is to understand how social determinants of health (Social and Community Context, Economic Stability, Education Access and Quality, Health Care Access and Quality and Neighborhood and Built Environment) impacts prenatal dental care. This data will help identify barriers and disparities in access to prenatal dental care, inform targeted interventions to improve oral health outcomes for pregnant individuals and their offspring, and guide policy recommendations to enhance equitable healthcare access.Item Embargo MOVEMENT AND HABITAT USE OF A RESIDENT KING RAIL (Rallus elegans) POPULATION AMONG MANAGED IMPOUNDMENTS AND TIDAL MARSHES IN COASTAL SOUTH CAROLINA(East Carolina University, May 2025) Linke, Megan MichelleFreshwater wetlands are important habitat for a variety of waterbirds. However, inland wetlands have sustained significant losses due to marsh drainage and land conversion for agriculture and human development. This loss of habitat has led to declining populations throughout the Eastern United States for an obligate freshwater marsh specialist, the king rail (Rallus elegans). Loss of inland habitat has pushed king rail populations toward the coastal margins of their range, where they are increasingly exposed to climate change related assaults such as hurricanes and associated storm surge, tidal inundation, and saltwater intrusion. To support habitat restoration and management efforts for a coastal king rail population, I investigated king rail movements and habitat use across a series of natural and impounded wetlands, interspersed with riparian forest along a tidally influenced river in coastal South Carolina. Some impoundments were managed for wintering waterfowl by planting fields with Carolina Gold rice during the king rail breeding season. Using VHF radiotelemetry, 13 king rails were tracked over the course of this two-year study generating Autocorrelated Kernel Density Estimated (AKDEc) home ranges. Breeding season 95% AKDEc was 5.1 ± 2.4 ha with a core 50% AKDEc of 1.1 ± 0.5 ha. Non-breeding season 95% AKDEc was 4.0 ± 2.3 ha with a core home range of 1.0 ± 0.5 ha. No significant difference was observed between the size of the breeding and non-breeding home ranges, and the size of home ranges observed here were smaller than other reported home ranges, suggesting the possibility of high prey and/or high king rail densities. Shifts to distinct non-breeding home ranges were observed between May and July and were preceded by either the successful molting of an instrumented individual’s chicks, nest failure, or habitat disturbance through management within the home range. Individuals that were heavier upon capture were more likely to occupy the same home range throughout the year, staying on the breeding home range for the non-breeding season. During the 2024 breeding season, king rails used habitat closer to channels in areas with higher percentages of marsh vegetation. Dominant vegetation types included giant cutgrass (Zizaniopsis miliacea), smartweeds (Polygonum ssp.), sedges (Carex ssp.), coffeeweed (Sesbania herbacea), and primrose willow (Ludwigia peploides). King rails continued to use areas within their established home range if it had been treated with herbicide, as the dead vegetation likely provided the same habitat characteristics as it did when it was alive, and the rails were already established in the area. A stem count measurement revealed that king rails avoided the densest marsh vegetation and selected areas with more moderate densities. My results suggest that when managing impoundments for king rails alongside wintering waterfowl, it is important to provide vegetation along channels and irrigation ditches within rice fields to provide cover while king rails forage along the edges of the channels. The removal of vegetation when planting rice and during dike construction reduced the likelihood for king rails to stay on their breeding territories and resulted in some individuals fleeing the area. Management techniques to control the density of emergent vegetation such as prescribed fire may be useful to keep a moderate density on the landscape.Item Embargo Ecdysone Receptor modulates EGFR signaling for timely cyst packaging in Drosophila germ cells(East Carolina University, May 2025) Swain, Lindsay AdelleOocytes are packaged into ovarian follicles, each containing a maturing germ cell surrounded by a layer of somatic cells that secrete protective eggshell and chorion proteins. This conserved arrangement is essential for proper oocyte development and reproductive success. Continuous coordination and bi-directional signaling from somatic cells to germ cells is necessary for proper oocyte packaging. In Drosophila, although germ cell packaging was presumed to be largely controlled by somatic follicle cells enveloping passive germ cells, recent studies suggest that germ cells themselves produce motor forces that drive somatic encapsulation. Here, in support of this hypothesis, we present data suggesting that cyst encapsulation is dependent upon Ecdysone Receptor, a steroid hormone receptor known to control multiple aspects of oogenesis. Using tools to deplete EcR levels or block transcriptional activation specifically in the germline, we show that germline-autonomous EcR is necessary for the timing of cyst encapsulation. In the absence of EcR, germ cell encapsulation is slowed, resulting in increased incidence of cyst collision events in the germarium. EcR facilitates germ cell cyst encapsulation by signaling somatic cells to promote proliferation, organize escort cell projections, and intercalate stalk cells. We show that an increase in germ cell cortical contractions is sufficient to rescue cyst collisions. Finally, we conclude that EcR is likely acting with EGFR signaling to modulate escort cell activity. Overall, these data suggest that in addition to its well-characterized roles in somatic follicle cells, EcR is necessary in the germline to promote timely ovarian follicle assembly and development.Item Open Access Habitat selection, prescribed burn effects, and passive monitoring of Eastern black rails (Laterallus jamaicensis jamaicensis) in North Carolina(East Carolina University, December 2024) Kephart, Bailey A.Eastern black rail (Laterallus jamaicensis jamaicensis) populations have experienced rapid declines from habitat loss exacerbated by sea level rise and human development. The suitable habitat for this species is broadly described as marsh containing dense, herbaceous vegetation at high elevations with little to no standing water (~ 0-5 cm). However, habitat characteristics, such as vegetation species composition and hydrology, vary by location where black rails are found. In North Carolina, birds are concentrated in only a few areas of coastal marsh that are subject to erosion and likely to be lost within the century. Additionally, this species’ behavior and phenology is poorly understood. As the black rail faces threats of extinction, it has become increasingly vital to better understand the species’ activity patterns and identify viable management strategies to facilitate marsh migration away from the shoreline and preserve existing populations. Prescribed fire may be a useful tool for maintaining patches at an early-successional state and promoting quality habitat, but its effectiveness has not yet been assessed for the mid-Atlantic coastal plain. In Chapter 1, I identify and discuss characteristics of suitable habitat as they relate to burn management and the probability of black rail occupancy. I conducted callback surveys during the breeding season at sites that were either burned in mid-winter or left unburned in 2022. I created occupancy models to identify predictors of suitable habitat. In separate analyses, I directly compared these variables among sites left unburned, within one year of burn, and one-year post-burn. These results were then integrated to assess the effects of prescribed fire management on black rail habitat and the effect of burn status on habitat use. In Chapter 2, I uncover temporal patterns of vocal activity and propose a phenological timeline for black rails in coastal North Carolina. I used passive monitoring techniques to document aspects of phenology and behavior during the breeding season. During 2023, I deployed recording units and monitored water levels at Cedar Island NWR. Lunar illumination values were extracted for each hour that was recorded. Counts of verified detections (3-second segments of recordings of black rail vocalizations) were then incorporated into models that identified temporal predictors of 1) vocalization probability and 2) counts of call segments during active periods. Predictors of vocalization probability were assessed separately from estimated counts. Over the 2-year period, I deployed motion-activated trail cameras at locations where black rails were detected to uncover evidence of phenology and breeding activity. I captured evidence of a presumed immature black rail after the completion of its juvenile molt. This 2022 observation is the first record of confirmed breeding by black rails in North Carolina in over 120 years. Results of this project provide insights into habitat preferences, vocalization patterns, and breeding phenology of Eastern black rails in North Carolina.Item Open Access Nutrient effects on soil fungal communities in a coastal plain wetland ecosystem(East Carolina University, December 2024) Siebor, Scott WyattAnthropogenic influences such as urbanization and intensive agriculture practices modify nutrient and water cycles in significant ways. These activities can cause disruptions to microbial mutualisms, especially in low-nutrient wetland ecosystems. Due to ongoing alterations to nutrient and moisture variations, the composition of fungal microbe communities may be shifting in unexpected ways. In this study, I investigated how long-term fertilization, and hydrologic alterations affect soil fungal microbial communities in a historically low-nutrient coastal plain wetland. I hypothesized that long-term fertilization and varying water conditions through ditching influenced patterns in fungal communities to different degrees. I tested this hypothesis at a long term nutrient addition (N-P-K fertilizers) and disturbance (mowing) experiment (established in 2003) located at East Carolina University's West Research Campus in Greenville, North Carolina. I specifically examined fungal microbe communities (based on amplicon sequencing of the internal transcribed spacer (ITS) region between the small and large subunits of the ribosomal RNA gene) in mowed plots undergoing and not undergoing nutrient enrichment. Results revealed that nutrient enrichment and ditch effects influenced fungal community composition, with community evenness being higher in wetter, unfertilized soils than drier fertilized soils over time. Additionally, soil treatments were more strongly associated with specific subsets of less abundant fungal families.Item Embargo The Influence of a Digital Weight Loss Intervention on Blood Pressure Outcomes(East Carolina University, July 2024) Maloney, Kallie R. StephensCardiovascular disease remains a highly prevalent disease that is consistently among the leading cause of death for Americans (Ahmad, 2023). Managing one’s blood pressure is a prominent recommendation in preventing heart disease (Olsen et al., 2016), but over 45% of the US population is classified as having hypertension (Benjamin et al. 2019). While the treatment recommendations for hypertension depend on hypertensive stage and ASCVD risk, lifestyle modification is recommended for all. Typical lifestyle changes include weight loss, diet modification, and increased physical activity (Whelton et al., 2018). Lifestyle intervention is not without challenge, however, with physician time constraints consistently emerging as a barrier to implementation. Digital health provides an avenue for assistance with lifestyle modification, something recognized by a majority of physicians (AMA, 2022). Digital health has shown to be effective in managing various physical health outcomes, including weight loss and diet modification. Little research, however, has focused on hypertension specific goals. Digital health also serves as an opportunity to reach underserved populations, such as those from rural or low-income environments. Preliminary research indicates that digital health interventions are feasible in these populations, but little research has assessed blood pressure outcomes. The aims of the present study, therefore, were to assess the influence of a commercially available digital health intervention for weight loss, Noom, on blood pressure related outcomes, determine what factors influence blood pressure, and determine if rurality and income environment influences rate of blood pressure decrease. It was hypothesized that systolic and diastolic blood pressure would decrease over the 16-week intervention. It was also hypothesized that weight, gender, and physical activity (steps) would be associated with blood pressure. Lastly, it was hypothesized that both rural users and users living in environments with a higher proportion of individuals from low income would display slower blood pressure decrease. User’s weekly blood pressure, weight, and steps were utilized for analysis. User zip codes were used to classify users as rural or urban using secondary Rural Urban Commuting Codes. Zip codes were also utilized to estimate the proportion of individuals from low-income households (<$25,000 annually) within a user’s zip code using 2019 IRS tax data. From there, conditional growth curve models were estimated to address all hypotheses. Systolic and diastolic blood pressure significantly decreased over the 16-week intervention. Baseline weight and physical activity were significantly associated with systolic blood pressure. Lastly, rurality status and income environment were not significantly associated with different systolic or diastolic blood pressure trajectories. While many findings were as expected, rurality and user’s environmental income not significantly slowing rates of blood pressure decrease were surprising. It is possible that the present sample of Noom users do not adequately reflect the average rural individual, or individuals from low income environments. Specifically, the cost associated with Noom and the voluntary nature of inputting blood pressure could have contributed to a sample that was more motivated for blood pressure change.Item Embargo Routes of transmission of gut bacteria in R. imitator tadpoles in an experimental context(East Carolina University, July 2024) Belduque Correa, NataliaThe gut microbiota provides important capacities for the host, and the relationship between host-microbiome could affect, bidirectionally, different traits of the history life. Therefore, it is important to know how the microenvironment of living beings is shaped and what factors influence the community and composition of bacteria. The mother–to–offspring microbiota transmission has been extensively studied in vertebrates, focusing on human primates. In amphibians, the understanding of diversity and community of bacteria has been biased to skin microbiome. In this research, we study whether R. imitator mothers, a neotropical poison frog from Peru that display prolonged parental care, are transferring microbes to tadpoles by feeding them with trophic eggs. We used 16S rRNA amplicon-sequencing of the gastrointestinal tract of captive R. imitator mothers and their tadpoles. The analyses showed that composition of microbes is different between parents and offspring. Also, the composition of gut bacteria of tadpoles included different bacterial phyla usually found in gastrointestinal tract of vertebrates. Although, phyla Bacteroidetes and Proteobacteria were also found in gut of fish food experiment, it was seen that the proportion of Proteobacteria are higher in tadpoles from switching and control experiments, which can suggest that the proportion of microbes that comes from the maternal diet is similar than the proportion that comes from the environment. This supports the assumption of mother’s role in parental care is not limited, exclusively, to provide nutrient resources.Item Embargo Nitrogen and Phosphorus Remediation in Mesocosm Floating Treatment Wetlands receiving Municipal and Aquacultural Wastewater(East Carolina University, July 2024) Barnes, Matthew ThomasAs populations shift and grow throughout the world, the amount of organic waste generated directly and indirectly by humans has increased. Wastewater treatment operations play a key role in helping to keep our natural surrounding water bodies clean by removing nitrogen (N), phosphorus (P), and other contaminants of concern from wastewater. Floating treatment wetlands (FTWs) may offer a low-cost energy efficient supplemental treatment option to existing wastewater treatment operations by removing nutrients from wastewater through plant uptake, adsorption, and microbial processes. The purpose of the study was to determine the N and P reduction capabilities of mesocosm FTWs from both domestic and aquaculture wastewater. The extent to which FTW plant species diversity (monoculture vs polyculture) influenced N and P removal was investigated, and the N and P removal dynamics throughout the growing season were characterized. Water samples and plant measurements were collected every week over the course of 14- and 10-week trials during 2022 and 2023, respectively. Mesocosm FTWs were located at the Greenville Utilities Commission Wastewater Treatment Plant for domestic wastewater research and the Marine Aquaculture Research Center of North Carolina State University for aquaculture wastewater research. For both trial 2 studies of the MARC and GUC-WWTP, there were no statistically significant differences among treatments of nitrogen (N) or phosphorus (P) removal. However, for all trials N was removed more effectively than P. The GUC-WWTP had removal ranges of 0.12 to 0.32g/m2/day for N and 0.01 to 0.08 g/m2/day for P. The MARC trial 1 had removal ranges of 0.49 to 0.95 g/m2/day for N and 0.02 to 0.04 g/m2/day for P whereas the trial 2 study has removal ranges of 0.21 to 0.43 g/m2/day for N and 0.004 to 0.02 g/m2/day for P. Overall, the late summer growing season (MARC 1) had higher nitrogen removal rates compared to late spring and early summer (MARC 2). For the GUC-WWTP, there was more phosphorus removed compared to the MARC, for trial 2. Yet all achieved positive removal rates in summation of both locations. The purpose of this study was to characterize the N and P removal efficacies of monoculture vs mixed species plantings of floating treatment wetlands from wastewater. Additional research into native wetland plant species and their nutrient removal potential could aid in finding alternatives or supplements to high energy-dependent treatment methods at wastewater treatment facilities. Future studies should investigate how water hydraulic retention times, continuous flow vs batch systems, and seasonal plant growth characteristics impact nutrient removal efficacies. Other studies into plants with more P removal efficacy and plants with more nutrient specific uptake rates could be effective depending on the nutrients being removed.Item Embargo Identification of a novel autonomous role for Ecdysone Receptor during Drosophila ovarian germ cell differentiation.(East Carolina University, July 2024) Jung, Lauren ElizabethA tightly regulated network of signals coordinates the division of Drosophila germline stem cells (GSCs) and differentiation of their daughter cells to produce viable oocytes. The steroid hormone, ecdysone, is known to promote GSC self-renewal; however, due to lack of compatible genetic tools, potential autonomous roles in the germline have not been fully evaluated. Ecdysone elicits a diverse array of transcriptional responses by binding to a heterodimeric complex composed of Ecdysone Receptor (EcR) and Ultraspiracle (Usp). To elucidate whether EcR facilitates autonomous reception of ecdysone in the germline, we built germline-compatible genetic tools to manipulate EcR levels or activity. Depleting EcR mRNA or loss of EcR ligand binding in the germline caused a loss of GSCs over time, demonstrating that it is necessary for GSC self-renewal. Further, over-expressing either EcR.A or EcR.B1 isoforms resulted in undifferentiated germ cell tumors and decreased numbers of GSCs. This is a ligand-dependent function of EcR, as over-expression of EcR that cannot bind ecdysone suppressed tumor development. By restricting over-expression to later stages of the germline, we found that EcR is sufficient to induce tumors only when over-expressed in the GSCs and early daughter cells. In tumorous ovaries, stem-like cells were also identified outside of their normal stem cell niche, suggesting that these cells remain competent to respond to BMP signals. Using single-cell RNA-sequencing to compare EcR over-expressing and Tkv over-expressing ovaries, we found remarkably similar transcriptomes in heterogenous populations of germ cells. These data suggest that EcR promotes differentiation of germ cells by directly regulating components of the BMP signaling pathway and that low levels of EcR expressed in wildtype GSCs are sufficient for ligand-dependent activation of an ecdysone-responsive transcriptional program. Altogether these data reveal a novel, autonomous role of EcR in GSC maintenance and regulation of differentiating daughter cells.Item Embargo Examining Microbial Transfer Between Human and Non-Human Primates Using a One Health Approach(East Carolina University, July 2024) Smith, DejahThe health of humans, wildlife, plants, and their environments are interconnected, and this concept is fundamental promoting public health and conservation goals globally. The One Health approach defines the relationship of humans, plants, animals and our shared environments, positioning health at the core. This approach aids conservation management as facilities can look at health holistically, focusing not only on the health of the captive populations but also of the staff, volunteers, and the public. This is particularly important for the conservation of endangered species, such as lemurs. In Madagascar, lemur populations represent 20% of the worlds primate species and are endangered due to habitat fragmentation, deforestation, and anthropogenic factors. Captivity gives endangered populations a chance at rehabilitation and humans the opportunity to observe and learn from their behaviors, physiology, and development. Although captivity holds many benefits, we must think about how factors introduced by conservatory settings influence the health of captive animals and the humans who interact with them. Past studies have documented the transfer of microbes is documented to occur between humans and their pets. In this study, I examined the extent to which microbiome exchange occurred between human and non-human primates observed at the Duke Lemur Center (DLC) in Durham, North Carolina, USA. I hypothesized that (i) lemurs exposed to human microbiomes frequently (i.e., daily) and housed in captive only settings will exhibit altered gut microbial compositions and higher microbial diversity of human associated taxa than free-ranging lemurs that interact rarely with humans and maintain spatial proximity or no human contact. I also hypothesized that (ii) humans working in Duke Lemur Center departments with high levels of contact between NHPs will harbor an altered skin microbiome, composed of bacterial taxa associated with lemur species. To test this hypothesis, I collected fecal samples from four lemur populations, which included blue-eyed black lemurs (Eulemur flavifrons), Coquerel’s sifakas (Propithecus coquereli), crowned lemurs (Eulemur coronatus), and ring-tailed lemurs (Lemur catta). This research was approved by the East Carolina University’s Institutional Animal Care and Use Committee (IACUC) protocol number: AUP#P110. Human forehead and hand skin swab samples from 13 interns over five timepoints were provided voluntarily from the DLC summer interns who worked in various departments across the facility. These protocols were approved by East Carolina University’s Intuitional Review Board (#UMCIRB 22-000802). The gradient of lemur-human interaction intensity across DLC department was done to ensure a variety of frequency of interaction with the lemur populations. There were seven husbandry interns, four research interns, one education intern and one paleo-primatology intern. I extracted genomic DNA from human skin and lemur fecal samples and conducted 16S rRNA gene PacBio HiFi long-read sequencing. Results showed that human-associated taxa were observed to a greater extent in the gut microbiomes of enclosed lemurs where there was more frequent contact with humans. Variation in diversity was observed in the free-ranging, terrestrial population, Lemur catta, who I observed in the field as approaching gated areas and trails that people often occupied. In human forehead samples, taxa associated with the phylum Bacteroides were observed. Both lemur fecal samples and human skin samples harbored Bacteroides, demonstrating the presence of non-endemic microbes in lemur guts. In humans, this revealed that animal caretakers can exhibit altered microbial diversity due to contact intensity with lemurs since taxa from the Bacteroides phylum were observed in high relative abundance on the human skin microbiome of the animal husbandry interns only, which is contrary to past studies. Understanding the mechanisms of microbiome transmissions among humans and non-human primates is especially important for the conservation of captive non-human primates and the health of the humans managing captive populations. Transmission of microbes among humans and non-human primates can provide key information on how to best serve these captive populations and manage facilities to ensure the best outcomes for rehabilitation and conservation of species.Item Embargo Chronic Low Dose Toxicity of Acrolein in C. elegans Mitochondria(East Carolina University, July 2024) Yan, LisaAcrolein (ACR) is a chemical of interest in the pathology and progression of mitochondrial disease and disorder. Chemical exposure to ACR may occur exogenously due to the use of herbicides or burning of fossil fuels, or endogenously as a by-product from various metabolic pathways. Several studies focus on the impact of high dose acute exposure of ACR with various model organisms, including live rats and mice, human spermatozoa, and several cell culture models. These models do not address the consequences of long term or chronic, low dose exposures and impacts on progeny. For these reasons, the model C. elegans was ideal because of its established genome, ability to self-fertilize or sexually reproduce, ability to yield three hundred progenies in its lifetime, and its short and understood lifespan. We hypothesized that C. elegans would experience mitochondrial dysfunction when chronically exposed to low doses of ACR, particularly with the 15 μM concentration. Based on previous research indicating another electrophilic aldehyde, 4-hydroxynonenal (4-HNE) inhibits SDH activity by forming a protein adduct with the redox subunit of SDH, we predicted that ACR may inhibit complex II (SDH) activity through protein adduct formation. We also predicted that ACR may also decrease lifespan and fecundity across generations of nematodes. To test our hypothesis and aims, we utilized C. elegans in experiments incorporating lifespan and fecundity assays, performed a histochemical stain to qualitatively measure SDH activity, used RT-PCR targeting specific genes related to oxidative stress and SDHA-1, and attempted to isolate mitochondria to perform SDH activity assays measured with spectrophotometry. Our results from the lifespan and fecundity assays suggested that low dose, chronic ACR exposure may decrease lifespan and fecundity in C. elegans. We also learned that C. elegans will attempt to leave the agar plate at ACR concentrations of 75 μM and 100 μM. Our RT-PCR results showed an upregulation in SDHA-1 and SOD-3 transcription in C. elegans after a 24-hour exposure to 100 μM ACR. These results suggest that higher doses of ACR may cause oxidative stress or damage to the mitochondria and SDH. Further experimentation may help better determine the interaction between ACR and the flavonoid subunit of SDH.Item Embargo Brucella spp. FtrB copper binding thermodynamics and iron oxidase activity(East Carolina University, July 2024) Hart, Kai AlexanderFtrABCD is a four-component iron uptake system found in several Gram-negative bacteria, including Brucella spp. This four-component transporter consists of two soluble periplasmic subunits (FtrA and FtrB), a putative membrane embedded terminal electron acceptor (FtrD), and a membrane spanning iron permease (FtrC). Based on the evolutionary relationship and sequence homology between FtrC and eukaryotic Ftr1p, this bacterial permease is predicted to be an oxidase dependent Fe2+ transporter. Unlike the eukaryotic Ftr1p permease, which can only function when co-expressed with the multicopper oxidase Fet3p, FtrC does not co-express homologs of any known ferrous oxidase. However, based on its evolutionary relationship with cupredoxins and ferroxidases, periplasmic FtrB is proposed to bind and oxidize Fe2+ during its transport through FtrC. This oxidase property of FtrB can be achieved if it binds a single copper ion using conserved D118 and H121 residues. This non-classical copper ion binding site in FtrB and its ability to form the active enzyme substrate complex (Cu2+ -FtrB-Fe2+) producing the ferrous oxidase function has not been verified experimentally, creating a knowledge gap in the bacterial iron uptake and redox enzyme literature. Data presented in this thesis demonstrates that a) recombinant wild-type Brucella FtrB can form a predominantly β-sheet containing structure; b) the ability of this protein to form this expected native secondary structure and coordinate Cu2+ (Kd = 3.0 ± 1.0 μM in 50 mM bis-tris, 100 mM NaCl, pH 7.3) is dependent on the presence of the conserved residue D118; and c) the recombinant wild-type FtrB can effectively oxidize Fe2+ at pH 7.3 under in vitro conditions. The results described in this thesis are significant as they demonstrate FtrB as a novel ferrous oxidase and open future research opportunities to investigate the importance of this Fe2+ oxidation during iron transport through FtrABCD under in vivo conditions.Item Open Access Biogeographical insights into the aquatic metal and antibiotic resistome(East Carolina University, July 2024) Joyner, CoryAntibiotic resistance in environmental microbiota is a naturally occurring, yet looming threat to global society and is estimated to contribute to over 10 million deaths annually by 2050. While human-derived contamination from industrial activity and wastewater worsens this, other environmental stressors like heavy metals and the genes conferring their resistance (MRGs) within microbes may enhance antibiotic resistance genes (ARGs). Ultimately, this leads to their persistence due to shared export mechanisms and coupled transcriptional regulation. However, the occurrence and relative abundance of MRGs and ARGs remains poorly understood across aquatic ecosystems and geographies. Here, we hypothesized that estuary ecosystems and highly industrialized countries would harbor the most abundant and diverse repertoire of ARGs and MRGs within its waters, showing ecosystem and geographic differences in resistome composition that could reflect the extent of human contamination within. 57 aquatic shotgun sequencing projects were downloaded from MG-RAST and the SRA spanning global waters and their nucleotide sequences annotated using the two-step ARGs-oap pipeline built from the Structured Antibiotic Resistance Gene Database (SARG), the Bacterial Metals and Biocides Database (BacMet), and a custom mobile genetic element (MGE) database curated to include plasmid, transposase, and integrase sequences. To determine the most abundant and frequently co-occurring ARG and MRG types, the relative abundances were stratified and plotted by ecosystem, country, continent, and hemisphere, while pairwise spearman correlation matrices were constructed to assess co-occurrence patterns and the results visualized with network analysis. Lastly, co-resistance was studied further within the environmental isolate Arthrobacter aurescens TC1 using chromium-enriched and marine salt antibiotic Kirby-Bauer disk diffusion assays. It was found that estuaries were more likely to have higher abundances of resistance genes for tetracycline resistance compared to marine environments, but held elevated concentrations of zinc, chromium, iron, arsenic and lead compared to freshwater and marine environments. Kenya carried an elevated abundance of tetracycline and sulfonamide resistance genes compared to both the industrialized nations of the USA and China, but no differences in MRG abundance were observed. Network analysis revealed that some multidrug efflux genes were highly correlated to MRGs for copper resistance, but genes conferring resistance to biocides and multi-metals may play an undermined role within aquatic ecosystems due to their prominence. Laboratory studies revealed that low concentrations of Cr(VI) under estuarine salinity conditions improved the resistance of known metal-resistant A. aurescens TC1 to many antibiotics, including bacitracin. These results suggest that estuaries and freshwater environments are more at-risk environments for ARG/MRG co-occurrence compared to marine systems, and Lake Victoria in Kenya is at a risk for ARG/MRG co-occurrence and possibly co-resistance, highlighting it as a promising location for future studies.Item Open Access Prevalence and environmental determinants of Black Gill Disease in Penaeid shrimp within Pamlico sound.(East Carolina University, July 2024) Gabriel, Chloe ElizabethPenaeid shrimp are one of the most ecologically and economically important species in North Carolina estuaries as well as the whole Southeastern United States. These species have historically brought in large amounts of revenue to North Carolina; in 2022, the shrimp fishery brought in over 10 million dollars to the State. Penaeid shrimp also serve other fisheries by being an important prey item for economically and ecologically important fish species like Flounder and Drum. As penaeid shrimp are ecologically and economically important, it is imperative that any threats to local shrimp populations are tracked. A recent threat to penaeid shrimp in the Southeast U.S. is a parasitic illness called Black Gill Disease. In the southeastern U.S., Black Gill Disease is the result of melanization and inflammation of shrimp gill tissue as a result of an infection by a parasitic ciliate species Hyalophysa lynni. Hyalophysa lynni is assumed to be native to the Gulf of Mexico, but has expanded its range northward, potentially facilitated by climate change, where warming temperatures have created more favorable conditions at its northernmost ranges. With the recent appearance of black gill disease in North Carolina, detection methods must be accurate to avoid underestimation of black gill impacts on the shrimp population and increase understanding of the disease impacts in this area. In chapter 1 of this thesis, two methods of detection (microscopy and genetics) are compared to determine the most efficient and accurate approach to identify infected shrimp. These methods are also discussed within the context of the current “on the boat” monitoring efforts conducted by the North Carolina Division of Marine Fisheries during the P195 survey. I hypothesized that a combination of microscopy and molecular techniques would provide the most accurate estimation of black gill disease prevalence. I found microscopy to be both efficient and accurate in detecting all stages of infections as well as being a more affordable and time efficient detection method when compared to PCR. Statistical analysis supported that there was not much difference between detection methods in their abilities to quantify black gill disease prevalence. Based on sequence data, my work successfully confirmed the cause of sBG in Pamlico Sound to be the ciliate Hyalophysa lynni, the same species that is suspected to be the cause of sBG in other states. Moreover, my data showed that microscopy and PCR results strongly aligned with one another (97% agreement). In Chapter 1, I also applied microscopy-based prevalence data to describe spatial and temporal patterns of black gill disease in Pamlico Sound as observed throughout the project. I also designed a descriptive black gill severity scale, which can be used to assist in future projects and aid fishery agencies in their monitoring efforts by standardizing severity scales. By determining the most accurate and efficient black gill disease detection method, projects and fishery-based agencies can apply these methods to increase their understanding and accurate monitoring of the disease. In chapter 2 of my thesis, I aimed to fill a knowledge gap regarding how white shrimp Penaeus setiferus respond to lower salinities and abrupt declines in salinity. To do so, I conducted an experiment exposing shrimp to varying low salinities and monitoring them for common stress behaviors and survival. Penaeus setiferus (commonly referred to as “white shrimp”) occurs in the Atlantic from the Chesapeake Bay to the Gulf of Mexico and is a common fishery for many of these states. This species of Penaeid shrimp moves between brackish estuaries and ocean waters at specific life stages. Due to this movement, it is generally assumed that Penaeus setiferus has a relatively broad salinity tolerance range, though there is a lack of literature describing how well it survives and responds to low salinities. My experiment exposed white shrimp from Eastern NC to three low-end salinities (2ppt, 5ppt, 15ppt) to determine if there were significant survival and behavior changes. I hypothesized that lower salinities would result in higher mortality rates and increased stress behaviors. I observed that shrimp placed in 15 ppt treatments showed the least mortality and stress behaviors while shrimp placed in 2 ppt showed the highest mortality and stress behaviors. These results could be important for understanding changing salinity levels in estuaries as a result of climate change and increases in episodic storm events, and the ways that species may respond to these rapid changes.Item Open Access Investigating the Role of the Antimicrobial Peptide TTO-53 in the Disruption of Pseudomonas aeruginosa Biofilm(East Carolina University, July 2024) Tilley, HannahPseudomonas aeruginosa infections are commonly acquired in hospital settings and are oftentimes severe and deadly. P. aeruginosa is known for its resistance to a wide range of antibiotics due to the low permeability of its outer membrane, possession of various efflux pumps and modifying enzymes, and its ability to form biofilms. Biofilms are aggregates of bacterial cells enclosed in an extracellular matrix. This extracellular matrix is composed of an assortment of extracellular polymeric substances (EPS’s) that can interact with and influence the rate of transport of antimicrobial agents. Previous research using a synthetic antimicrobial peptide made of unnatural amino acids named TTO-53 has shown its ability to disrupt pre-established P. aeruginosa biofilm. The goal of this study is to investigate how this disruption is taking place and the influence of this peptide on the efficacy of antibiotics in killing P. aeruginosa. Due to the role of quorum sensing, a communication system between cells in biofilm, in the maintenance of biofilms, we hypothesized that exposing P. aeruginosa to TTO-53 causes disruption of quorum sensing resulting in dispersion of pre-established biofilm. To test this hypothesis, we used quantitative real-time PCR (qRT-PCR) to determine if there was differential expression in genes within various quorum sensing pathways. Results from qRT-PCR showed that exposing overnight cultures to TTO-53 did not cause differential expression in genes within the LasR, PQS, and RhlI/RhlR quorum sensing pathways. To investigate the influence of TTO-53 on the efficacy of various antibiotics, we exposed cultures of PAO1 to varying concentrations of each antibiotic and compared the colony-forming units per milliliter (cfu/mL) of those cultures to the cfu/mL of cultures treated with those same concentrations of each antibiotic and TTO-53. These results indicated TTO-53 increases the efficacy of Chloramphenicol and Tazobactam/Piperacillin, while decreasing the efficacy of Tobramycin. This study revealed that an hour-long exposure to TTO-53 does not impact P. aeruginosa quorum sensing pathways and the combination treatment of TTO-53 has a synergistic effect with some antibiotics while inhibiting others.Item Open Access Investigating the Role of Viral Proteins in COVID-19 Blood Clotting(East Carolina University, May 2024) Wright, RaegynnThere have been approximately 7 million deaths and 775,522,404 infections worldwide due to Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2). Many of the severe symptoms of COVID infection are associated with blood clot formation during infection. Post-Acute Sequel of COVID is also associated with symptoms that are hypothesized to be the result of clotting as well. Clot formation is a natural response to cellular damage within the vascular system and is facilitated by the release of a series of fiber-like structures known as Von Willebrand Factor (VWF). To prevent unnecessary clots from forming during vascular inflammation, an enzyme called ADAMTS-13 prevents the accumulation of VWF, in the absence of serious cell damage. This paper demonstrates that a viral protein capable of cleaving ADAMTS-13 may be responsible for the reduced levels of ADAMTS-13 noted in COVID-19 patients that is linked to increased clot formation during infection. Additionally, this interaction may provide insights into the rebound symptoms observed with the anti-COVID drug Paxlovid, a 3CL protease inhibitor, and provide clues to the nature of symptoms associated with, and potential diagnostic tools for long COVID.Item Open Access Parental Response to Breeding Pool Water Level Change in Ranitomeya imitator(East Carolina University, July 2024) Reynolds, MichaelFor parental care behavior to be effective in providing benefit to offspring requires the accurate fulfillment of offspring needs. Environmental changes alter the stressors placed on offspring, and parental care must change to meet these altered needs. Behavioral plasticity in parental care has been observed previously in amphibians, notably in males of the glass frog Hyalinobatrachium fleischmanni, which alter the lengths of time spent brooding eggs depending on ambient humidity and egg hydration levels. Dendrobatids, or poison dart frogs, often lay their eggs terrestrially and transport tadpoles to adequate aquatic habitats. Previous research has shown that parental investment in offspring care tends to increase in conjunction with smaller breeding pools. Among dendrobatids, the mimic poison dart frog Ranitomeya imitator is well known for raising its young in small phytotelmata, and for its unique reproductive system of monogamous biparental care of eggs and tadpoles. The small pools R. imitator raise their offspring in are ephemeral and small in volume, making them vulnerable to desiccation. Although R. imitator guard breeding pools from competitors and provide trophic eggs as food for tadpoles, there is no evidence that they provide water to tadpoles. To examine the behavioral responses of adults and the physiological responses of tadpoles of Ranitomeya imitator to water level changes in the breeding pool, we exposed 10 breeding pairs of frogs to three different treatment levels of water level change. Over the course of biweekly water changes, the fast treatment experienced a 2.5 ml water level decrease, the slow treatment decrease experienced a 1.25 ml water level decrease, and the control experienced no water level decrease. Adults were recorded attending to their tadpoles in artificial breeding pools, and the proportion of time each pair spent on different parental care behaviors was compared between groups. Soon after metamorphosis, metamorphs were weighed and measured. Parental care behaviors did not significantly differ in proportion between fast, slow, and control water level treatment groups. Significant differences were found between treatment groups in tadpole snout-vent length and weight in grams. Duration of the larval stage positively correlated with water level treatment, and the most significant differences between treatment groups were found between the fast and control treatment groups. These results suggest that when exposed to rapidly decreasing water levels R. imitator tadpoles are selected to optimize the tradeoff between body mass and larval stage duration. Plasticity in growth exhibited in tadpoles may be an adaptation to the ephemerality of the phytotelm environment, enabling tadpoles to escape from the aquatic environment more quickly. Future research into this system should examine the impacts of the tradeoff between body mass and larval stage duration on post-metamorphosis life history. Such research could have implications for the persistence of this species and other dendrobatid frogs under climate change.Item Open Access Hatch dates and habitat use of juvenile sheepshead Archosargus probatocephalus recruiting across a biogeographic divide in North Carolina.(East Carolina University, July 2024) Johnson, Maddie LeeThe goal of this study was to identify hatch dates of juvenile sheepshead (Archosargus probatocephalus) to confirm the spawning season for the species within North Carolina, and to identify potential spatial differences in early life history dynamics and habitat usage of juveniles. Spatial differences were compared across four locations to the North (Oregon Inlet) and South (Hatteras, Cedar Island, and Back Sound) of Cape Hatteras, a known biogeographical divide. Sheepshead are a coastal and estuarine species that are poorly understood in the region, especially concerning the juvenile stage. Sheepshead are managed by individual states and in North Carolina, a stock assessment is underway, and research on early life history dynamics was requested. The first objective of this study was to confirm the spawning season for sheepshead in North Carolina through a hatch date analysis. Transverse sections of sagittal otoliths were analyzed, and total age was used to identify hatch dates for juveniles in the four locations within North Carolina. The results determined that the general spawning season for sheepshead in North Carolina is April to May. Also, hatch dates varied spatially, especially between the Cape Hatteras breakpoint with fish from Oregon Inlet hatching later on average. The second objective was to determine fine-scale spatial differences in early life history dynamics across the Cape Hatteras breakpoint. Relationships between various important ages (total, pre-settlement, and post-settlement), timeframes (pelagic larval duration, juvenile stage), lengths (fork length and estimated size at settlement), and hatch dates were compared between locations. The results determined that early life history dynamics are variable across Cape Hatteras. Most notably, pelagic larval duration was longer for sheepshead collected north of Cape Hatteras. The final objective was to determine habitat utilization of juvenile sheepshead in Pamlico Sound. The utilization of various habitat types including seagrass beds, areas of oyster aquaculture, and unstructured sandy bottom habitats were compared. The results from this indicate that juvenile sheepshead have preferences for biogenic habitats, mainly areas of seagrass. The results from this study provide resource managers with more information on early life history dynamics of sheepshead in North Carolina. The confirmed spawning season can be further used as a baseline for additional research on reproductive biology of sheepshead. Additionally, this study provides methodology for any future research into a juvenile index of abundance, which can be used to create an index of annual recruitment. Finally, this study provides evidence of differences in early life history dynamics across Cape Hatteras and habitat preferences for the species.Item Open Access The Isolation and Characterization of Microbes from a Serpentinizing Environment(East Carolina University, July 2024) Sims, Devin LeeAn ancient geochemical process known as serpentinization is an environment that contains unique microbial communities and metabolisms. Serpentinization is a reaction between water and ferromagnesian minerals leading to the generation of serpentine minerals and products such as hydrogen, formate, acetate, and methane. The long history of this process along with its ability to produce sustainable energy makes serpentinization a strong candidate as a contributor to the emergence of life. While this process supplies necessary assets needed for microbial life, this process also creates extreme conditions that make life challenging for microorganisms. Serpentinizing environments present challenges such as high pH (>9), lack of inorganic carbon, and reducing conditions (low redox potential). These challenges existing in serpentinizing environments could be a major factor for metabolic influence on the evolution of microbes. Serpentinization is a widespread event occurring in a number of locations across the globe. Interestingly, recent studies using imaging technology reveal that this very exact process may in fact also occur on Mars. Many sites of serpentinization are relatively similar in their geochemical composition. However, among distinct locations, components involved and produced during serpentinization tend to vary in flux and quantity. This has been shown to have an effect on the microbial diversity and microbial abundance seen at varying sites. In this study microbes were cultivated and isolated from rock core samples extracted from the Coastal Range Ophiolite Microbial Observatory (CROMO). A genomic DNA extraction was performed to identify isolates using 16s rRNA sequencing. Metabolic and physiological assays were conducted to understand microbial response to specific environmental pressures as well as potential metabolic pathways utilized. This investigation allowed us to discover the metabolic potential of microbes representative of a serpentinizing environment. As a result, this will allow us to make more accurate projections and notions about microbial communities inhabiting serpentinizing locations and the types of metabolisms needed to navigate this type of environment.