Emerging evidence suggests that the immune system is vulnerable to disruption in response to a wide variety of chemical contaminants; thus, there is a need to test chemicals for immunotoxicity. To understand how chemicals impact the ability of the immune system to ward off infection, a model system featuring fathead minnows (FHM, a common toxicological model) infected with Yersinia ruckeri (a bacteria) has been used. Using this model system, the impacts of chemicals on immune system function can be evaluated via pathogen resistance challenges, where a fish is infected with a pathogen and their ability to defend against the pathogen and survive infection is determined. However, the use of Y. ruckeri is unfavorable given that fish must be injected with Y. ruckeri to develop an infection, a process that is time consuming and inconsistent with natural routes of exposure. Thus, the goal of this project was to develop a new host-pathogen system for FHMs by identifying a pathogen that induces infection via immersion. To do this, the ability of three bacterial pathogens, Flavobacterium columnare, Aeromonas sobria and Aeromonas allosaccharophila, to infect FHMs via immersion was evaluated. Results of this study revealed that of the three bacterium evaluated, only F. columnare was capable of inducing an infection via immersion in FHMs and infection was only successful following a fin-clip procedure, in which a small portion of the caudal fin is removed. Overall, this result establishes the potential for a FHM-F. columnare model system for future use in immunotoxicity testing.

Nickel is the most prominent heavy metal in the effluents associated with crude oil extraction and production. Given that these effluents are released into the ocean, investigating the toxicity of nickel on marine life is pertinent. A current method for evaluating the toxicity of oil effluents is the sheepshead minnow larval growth and survival (LGS) test, which exposes larval fish to varying concentrations effluents or associated single chemicals over a 7-day period. However, current legislation, like the Frank E. Lautenberg Act, requires that animal testing be refined whenever possible to enhance animal welfare. The fish embryo toxicity (FET) test, which investigates chemical toxicity using fish embryos over a 7-day exposure period, is a potential alternative method capable of meeting legislative needs related to animal welfare. The objective of this study was to determine if a sheepshead minnow FET test is a viable replacement for the sheepshead minnow LGS test. To accomplish this, the results of sheepshead minnow LGS and FET tests using nickel were compared. The results of this study show that the LGS test is more sensitive than the FET test. In addition, evidence suggests that it may be possible to improve the sensitivity of the FET test by including sublethal metrics as FET test endpoints.

Bacillus anthracis is a bacterial pathogen that causes the often lethal disease anthrax. This research aims to characterize the role of potential virulence genes in Bacillus anthracis. Virulence is a pathogen’s ability to damage the host. Studying virulence allows us to understand infection mechanisms and develop novel ways to target pathogens. Previous work identified a collection of potential virulence mutants (Franks et al, 2014) each containing a genetic disruption that renders a gene non-functional. These mutants were pulled out in initial screenings but were never characterized further. We confirmed that one mutant, TN2, also exhibits decreased virulence in a Galleria mellonella survival assay. We know that TN2 has a disruption in a promoter region that we hypothesize controls two genes: a putative BNR repeat domain protein (TN2A) and a glycosyl-like 2 transferase family protein (TN2B). For my project, I attempted insertional mutagenesis to inactivate these genes with the goal of confirming that the genes are linked to virulence, rather than unintended mutations elsewhere in the genome. After successfully creating insertional mutant 2B, through the disruption of the TN2B gene, I am working to further characterize the mutant to determine its role in immune evasion. Specifically, I will compare the ability of the wild-type and mutants to survive exposure to various antimicrobial defenses conserved in humans and waxworms. This research could help identify a novel bacterial virulence factor and its potential mechanisms of action thus expanding our understanding of bacterial pathogenesis.

The gram-positive bacterium, Bacillus anthracis, is responsible for the deadly disease Anthrax. B. anthracis is dangerous due to virulence factors, or defenses the bacteria uses to infect a host. We hope to better understand how this bacterium interacts with its hosts by studying the genes necessary for virulence. Bacterial mutants, which have a change in their genetic sequence, sometimes show reduced ability to cause disease in a host. Studying these mutants helps us understand the bacteria’s infection method. Previously our lab created a library of mutants using a technique called transposon mutagenesis and then screened these transposon mutants for phenotypes linked to decreased virulence. This resulted in the identification of 11 transposon mutants that were less effective at causing disease in the nematode Caenorhabditis elegans (Franks et al.). While all 11 mutants could be interesting for further characterization, it is necessary to prioritize them as this is still too many to study. In this project, we tested these mutants using a second infection model, the caterpillar Galleria mellonella. G. mellonella is an ideal model due to its optimal size for injection, conserved innate immune defenses, and previous success as an infection model for B. anthracis (Malmquist et al.). We found that only one of these 11 mutants, TN2, had reduced virulence in both C. elegans and G. mellonella. Future research will focus on confirming the genetic change in this mutant and determining the mechanism by which it contributes to infection. This could lead to new antibiotic targets in the future.

Mercury (Hg) is released into the environment by coal-burning powerplants and artisanal gold mines. Aquatic bacteria then convert the inorganic mercury into highly toxic methyl mercury. Turtles acquire mercury through their diet, and it bioaccumulates throughout their long lifetime. Toenail clippings can be used to determine Hg concentrations in turtles. Toenail samples were collected from Trachemys scripta elegans (red-eared sliders) in the Brazos River near Granbury and the Clear Fork of the Trinity River as it flows through Fort Worth. All toenails were dried in a 60℃ oven and put into a direct Hg analyzer that uses thermal decomposition, gold amalgamation, and atomic absorption spectrometry to determine total Hg. Toenails from the Brazos river had significantly more Hg on average than those in the Clear Fork, 658.302µg/kg and 400.146µg/kg respectively. The results were unexpected as the Brazos river near Granbury is considered less polluted than the Clear Fork of the Trinity, which is supported by observations of insect larvae such as hellgrammites which were common in the Brazos but absent in the Clear Fork of the Trinity. Our hypothesis is that red-eared sliders in the Brazos are feeding at a higher trophic level than those in the Clear Fork. Fecal samples and a lack of invertebrate prey in the Clear Fork suggest red-eared sliders primarily feed on algae. In the Brazos river we observed several species of insect larvae underneath rocks and hypothesize that the red-eared sliders are feeding on this abundant food source. Mercury is known to biomagnify and therefore red-eared sliders in the Brazos are likely ingesting more mercury than those in the Clear Fork.

Alzheimer’s Disease (AD) and Traumatic Brain Injuries (TBI) are global societal problems affecting millions of people and costing billions of dollars per year.1,6 Hallmarks of AD include memory loss, cognitive decline, depression, and confusion due to unchecked inflammation in the brain caused by the overproduction of pro-inflammatory cytokines by the immune system.1,9,11 TBI occurs when a sudden trauma damages brain cells, which activate the immune response potentially leading to chronic inflammation and a multitude of symptoms affecting cognitive, somatic, and emotional processes.3,4,12 There is currently no cure for AD, nor is there an effective treatment for chronic inflammation caused by TBI. P2D Bioscience® has manufactured a series of drugs successfully targeting inflammation in a 3XTgAD mouse model.21 To understand the cellular mechanism of the novel drugs, we used SDS-PAGE electrophoresis and Western Blot analysis to investigate protein levels within the NFB pathway, which modulates inflammation. We monitored the inhibitor of NFb, IB, to determine whether the drugs were blocking the phosphorylation and degradation of IkBa and subsequently blocking the pro-inflammatory effects of activated NFB. We show that the drug is blocking the degradation of IB, and therefore the pro-inflammatory genes associated with the NFB pathway are not being transcribed. Increasing our understanding of the cellular mechanism of action is imperative for the progression of drug development because it can be used to evaluate potential side effects.

Alzheimer’s Disease (AD) is a neurodegenerative disease that primarily affects elderly populations. AD engenders memory loss and cognitive decline, and its prevalence is rapidly growing. It is estimated that 14 million Americans will have AD by the year 2050. Therefore, it is imperative for researchers to examine the underlying biological mechanisms responsible for AD. Previous research has demonstrated that chronic inflammation is linked to the hallmark AD pathology, amyloid beta (Aβ). Aβ is a protein that disrupts neuronal communication and increases the production of effector proteins called pro-inflammatory cytokines. Microglia function like immune cells in the brain, and when they are activated by inflammatory triggers, such as Aβ, they secrete pro-inflammatory cytokines. Although cytokine release is initially a healthy response, excess cytokine production is harmful to the brain and exacerbates AD pathologies. Prior research has demonstrated that pro-inflammatory cytokines are upregulated in the serum of AD patients. Therefore, cytokines are a crucial target for AD therapeutics.

The current experiment will examine the temporal inflammatory response of microglial cells following lipopolysaccharide (LPS) insult. LPS is a component of common bacteria and can induce inflammation in microglial cells. We will treat cells with several different concentrations of LPS and assess cytokine production at several different timepoints. To do this, we will collect cell supernatant (secretions) and measure multiple cytokines using an ultrasensitive electrochemiluminscent assay. Data collected from these experiments will be used in many future studies of potential therapeutics and dietary supplements. In fact, data from these experiments will be used by current and future departmental honors students to determine the optimal treatments and times for their experiments. This project is incredibly relevant because AD is currently the 6th leading cause of death in the United States. Data collected will help us pinpoint proper testing procedures for therapeutic compounds that are developed.

Cells use diverse mechanisms to prevent DNA damage and tumor formation. Two tumor suppressors employed in this effort are the focus of our study: breast cancer type 1 susceptibility protein (BRCA1) and BRCA-1-associated RING Domain protein 1 (BARD1). These two proteins form a complex that suppresses the generation of estrogen-derived free radicals. Inherited mutations in BRCA1 or BARD1 are associated with an increased risk of developing breast or ovarian cancer in humans. The model organism Caenorhabditis elegans possesses the orthologs BRC-1 and BRD-1 which can be readily mutated, offering an attractive model to study biochemical functions. However, it is unknown if BRC-1/BRD-1 also regulates the transcription of estrogen metabolism (cyp) genes to control the production of free radicals as noted for the human homologs. Utilizing gene expression analysis and estrogen exposure assays, this study demonstrates that BRC-1/BRD-1 has a conserved function of regulating cyp genes in C. elegans. However, our data also shows that BRC-1 and BRD-1 do not necessarily protect DNA from free radical damage upon estrogen exposure, despite its proven inhibition of cyp genes expression. Further investigation is required to determine the function of these cyp gene homologs in C. elegans. Our findings of this additional conserved function of the BRCA1/BARD1 homologs in C. elegans further validate its use as a model organism to better understand the myriad ways BRCA1/BARD1 protects the genome.

BRCA1 and p53 have been proven to interact in tumor suppressor pathways for hereditary breast and ovarian cancer. Finding the physical binding location associated with this interplay is important in assessing cancer risk and determining molecular details of the interaction. This project aimed to identify the protein binding region of p53 with the intrinsically disordered region of BRCA1. We cloned select regions of human BRCA1 and p53 protein into E. coli bacteria, then harvested and purified the proteins. A pull-down assay was performed to test binding affinity between a segment of p53 and two different length BRCA1 constructs. The assay showed that neither the construct that contained BRCA1 amino acids between 772-1126 nor the construct with amino acids between 896-1190 interacted with p53. This indicates that these amino acids alone are not sufficient for binding of p53 and BRCA1. Our results could also indicate that a third-party binding mediator is required in vivo. This information expands upon our knowledge of the p53 and BRCA1 binding interaction and can be used in a clinical setting to evaluate risk associated with mutations in the experimental regions.

Alzheimer’s Disease (AD) is a progressive neurodegenerative disease associated with old age and marked by deficits in memory and learning skills. AD pathology is characterized by amyloid-beta (AB) accumulation, which leads to plaque formation and ultimately neuronal death. Additionally, AB activates microglial cells, which function as an immune cell in the brain. Microglial cells secrete proteins that induce inflammation, known as pro-inflammatory cytokines. The chronic activation of pro-inflammatory cytokines engenders neuroinflammation and oxidative stress, which then further exacerbates AD pathologies. This project aims to study the effectiveness of cannabidiol (CBD) as a potential treatment for AD, due to its known anti-inflammatory properties. We will measure the inflammatory response of cultured BV2 immortalized mouse microglial cells following lipopolysaccharide (LPS) treatment. We will then include a CBD treatment to study its therapeutic capabilities in reducing inflammation. We hypothesize that treatment with CBD will decrease the pro-inflammatory cytokines TNF-alpha and IL-6 induced by LPS stimulation. We will perform enzyme-linked immunosorbent assays (ELISAs) to detect and quantify the cytokine levels.The overall goal of the research is to demonstrate the capacity of CBD to minimize the immunological mechanisms that drive AD pathologies. Our research will contribute to the understanding of the link between the immune system and central nervous system in AD development. AD is the sixth leading cause of death in America, but the availability of therapies is limited. CBD represents a natural and possible effective therapy for those suffering from Alzheimer’s disease, and our research will contribute to determining its efficacy.

Effect of Season, Body Size, and Sex on the Mercury Concentrations of Orb-Weaving Spiders
Cale Perry, Garrett Helburn, Olivia Eberwein, Madeline Hannappel, Matthew Chumchal, and Ray Drenner
Mercury (Hg) is an anthropogenic contaminants found in all aquatic ecosystems across the world. One of the methods to monitor levels of Hg contamination in an ecosystem is using sentinel organisms: abundant and widely distributed organisms within the food web that accumulate contaminants in body tissues without negative effects. Riparian spiders are a potential sentinel organism for the study of Hg contamination in aquatic ecosystems, as they accumulate mercury through the consumption of contaminated emergent aquatic insects. The present study will examine the effects of spider body size, sex, and season on 2 taxa of orb-weaving spiders [Family Araneidae: Larinioides sp., Metazygia sp.]. 575 orb-weaving spiders were collected from a boat dock on the South side of Eagle Mountain Lake, Texas, USA, from May to September 2019. The spiders were preserved in 95% ethanol and sorted based genera, month collected, sex, and size class. Size class was determined by measuring the spiders front left leg length (tibia + patella) and served as an indicator of body size. Mercury contamination will be analyzed through direct Hg analysis.

Plants with threatened habitats and fragmented populations may require repatriation efforts to maintain healthy populations. Populations of Sarracenia alata, the pale pitcher plant, are severely fragmented, and the species is near threatened. A complete understanding of its reproduction will be crucial in establishing and maintaining healthy populations. The goals of this study were to determine if 1) S. alata is capable of selfing (reproducing with pollen from the same individual); 2) S. alata is capable of autogamy (selfing without intervention); and 3) pollen load affects reproductive success. We used seed set to measure individual fitness. Thus, it was necessary to determine a reliable method of counting seeds. Two methods were examined, and these gave statistically similar results. We found that while S. alata is capable of selfing, it is not autogamous. Seed set was significantly higher in outcrossed individuals than in selfed individuals . In 2019, plants receiving supplemental pollen yielded more seeds than those in either the control group or a group in which pollinator access was restricted. During 2021 (a year with higher pollinator activity), there was no significant difference between the number of seeds produced by control plants and those receiving supplemental pollen. This study demonstrates the important role of pollinators in maintaining healthy populations in this system.

Due to habitat loss the Texas horned lizard (THL) (Phrynosoma cornutum) population has declined across its historic range. To date, reintroduction attempts for the species have been unsuccessful, calling into question the suitability of the habitat. Texas horned lizards require suitable thermal habitat to meet their thermoregulatory needs, because of this, understanding the thermal habitat requirements of THLs is important. While the critical temperature limits and preferred body temperatures of THLs are established from laboratory studies, thermal habitat preferences for THLs in the wild are poorly understood. The objective of this study was to determine thermal habitat preferences and home range sizes of reintroduced THLs at Mason Mountain WMA compared to a nearby natural population of THLs on the White Ranch. We also compare the thermal conditions of different microhabitats between the two sites. To compare thermal conditions between the two sites, we used thermal dataloggers to record the temperatures in different microhabitats throughout the day at each study site, then compared how much of the time these data loggers were within the lizard’s optimal temperature range between the two study sites. Home ranges were calculated for lizards from the two study sites and average home range sizes between the two study sites were compared for significant differences. The ground temperature selected by the lizards versus random points were compared between the two study sites. These findings will improve our understanding of THL thermal ecology and reintroduction requirements.

Migration, which is defined as the seasonal movement for survival or reproductive advantage such as access to resources, is a behavioral phenomenon exhibited by many species including the salmonid Oncorhynchus mykiss. More commonly known as rainbow trout, O. mykiss exists in two life histories: migrants (steelhead trout), and residents (rainbow trout). While there are many factors that contribute to this variation in migration behavior, one of the reasons is their genetic makeup since there is an apparent correlation between the migratory behavior of parents and their offspring. The primary objective of this research project is to identify single nucleotide polymorphisms (SNPs), or genetic differences, which are associated with migratory behavior in rainbow trout. To that end, I used whole genome sequence data from five migrant and five resident rainbow trout. These data were aligned to the trout genome and used to locate genetic differences between the two migratory types. Quantitative PCR (DMAS-qPCR) approaches were used to validate the SNPs and genotype them in a larger set of twenty-five migratory steelhead. Research findings exhibited that Sashin Lake is producing smolts (young migratory steelhead) that are successfully returning to the lake and reproducing at the end of their life cycle. Additionally, while there was not a significant difference seen in terms of marine survival between the sexes, females were more likely to migrate compared to their male counterparts due to the reproductive advantage and greater access to resources that migration offers. This data will support future studies observing trout migratory behavior with larger sample sizes and from different generations and settings and will benefit conservation studies regarding population decline in migratory species.

BRCA1 is a gene whose protein (also named BRCA1) is found throughout all human cells and engages in DNA repair, cell cycle regulation, gene transcription regulation, and apoptosis. However, mutations in BRCA1 typically confer a higher risk of cancer in estrogen-responsive tissues, including breast epithelial tissue. This increase in incidence of tissue-specific cancers is thought to be in part due to the role of BRCA1 in the estrogen response pathway and interaction with the estrogen receptor alpha (ERα). Previous studies identified possible regions of each protein involved in the binding interface between BRCA1 and ERα. Using these regions (amino acids 177-240 in BRCA1 and the ligand binding domain of ERα) as our constructs, our studies further analyzed the molecular details of this direct interaction and determined methods conducive to studying the BRCA1-ERα interaction. A pull down assay qualitatively confirmed binding between the constructs of BRCA1 and ERα. Data collected from NMR spectroscopy reaffirmed the direct interaction between BRCA1 and ERα first seen in the pull down assay and provided evidence demonstrating that the presence of estrogen in the samples increased binding affinity. Finally, fluorescence spectroscopy of quenching experiments confirmed the previous two results – that a direct interaction between the constructs of BRCA1 and ERα used occurs and the binding affinity increases in the presence of estrogen – and allowed us to describe the binding curve of the system being studied. The molecular details confirmed here provide further avenues of study, such as documenting variants of unknown significance or studying the role estrogen plays in the function of the BRCA1-ERα complex, which could lead to novel findings that expand our understanding of the role either protein plays in cancer development.

Alzheimer’s disease (AD) affects about 6 million Americans, and hallmark pathologies of AD include amyloid beta (Aβ), inflammation, and oxidative stress. Microglial cells (MGCs) are brain cells that function like immune cells, and they respond to Aβ by secreting pro-inflammatory cytokines. Cytokines induce inflammation at sites of infection, and Aβ continually increases inflammation, resulting in neuronal death. Inflammation is also connected to oxidative stress, and prior research has demonstrated that Nrf2 (a transcription factor) protects cells from oxidative stress by increasing antioxidant enzymes. We will test potential benefits of molecules with antioxidant capabilities, created by Dr. Green (TCU Chemistry), on inflammation and Nrf2 expression in MGCs. Previously, we demonstrated that these compounds, L2 and L4, are powerful antioxidants that protect MGCs from oxidative stress. Currently, we aim to study the effects of L2 and L4 on inflammation, Nrf2 expression and heme oxygenase-1 (antioxidant) production following an inflammatory insult. We will pre-treat MGCs with different concentrations of L2 and L4, and then stimulate MGCs with lipopolysaccharide (LPS), a bacterial mimetic. Subsequently, we will measure pro-inflammatory cytokines, Nrf2 expression and antioxidant response genes. Overall, it is crucial for researchers to investigate effective therapeutics that could relieve AD symptoms, such as antioxidant treatment.

Testing of chemicals that enter our waterways is necessary to keep marine environments healthy. The current method of toxicity testing is the larval growth and survival (LGS) test, which exposes larval fish to varying concentrations of an effluent or chemical. Given recent legislation that calls for improvements in the welfare of animals used in toxicity testing, there is a need to identify alternatives to the LGS test. In light of this, the objective of the current study was to determine whether toxicity tests featuring fish embryos or shrimp could be used in place of LGS tests.

To accomplish this, we compared the results of the standard LGS test using inland silverside larvae with the results from two alternative tests, a mysid (e.g., shrimp) test and an inland silverside fish embryo toxicity (FET) test. The results of this study show that both the mysid and FET tests are promising alternative testing methods to the LGS test. The adoption of either test type will meet legislative goals and improve the welfare of fish used in toxicity testing.

The Effect of Body Size on Mercury Concentration of Orb-Weaving Spiders (Araneidae) from the Clear Fork and West Fork of the Trinity River

Authors: Tyler Williams, Iris Schmeder, Morgan Capone, Matthew Chumchal, Andrew Todd, Ray Drenner, Cale Perry, Tori Martinez, Macyn Willingham, Robby Peterson, Chris Allender

Mercury (Hg) is a contaminant threatening all ecosystems. Inorganic Hg is released into the atmosphere from power plants and artisanal gold mines before being deposited over the landscape. Inorganic Hg deposited in the water can be converted by aquatic bacteria to methylmercury (MeHg). Methylmercury is one of the most toxic forms of Hg due to its capability of bioaccumulating within the tissues of organisms. Overexposure of methylmercury can cause damage to the nervous, genetic, and enzyme systems in the body, leading to a multitude of health complications. Evaluating the amount of Hg in an ecosystem, and thus the risk to organisms, is not straightforward. For example, the concentration of Hg in water or sediment may not be representative of aquatic organisms’ exposure to Hg because not all the Hg in water or sediment is bioavailable. As a result, scientists measure Hg concentrations in sentinels, defined as: an organism that can accumulate Hg within its tissue without significant adverse effects and serve as a representation of the level of Hg present within an ecosystem. Riparian spiders consume emerging aquatic insects and are therefore sentinels of Hg contamination in aquatic ecosystems. The objective of the study was to evaluate the concentration of total Hg in orb-weaving spiders (Family Araneidae) from the Clear Fork and West Fork of the Trinity River and determine how Hg concentration changes with spider body size. Spiders were preserved in 95% ethanol and body size was measured. Spiders were then dried and analyzed using a Direct Mercury Analyzer (DMA).

The reintroduction of captive-bred animals has been increasingly utilized for the conservation of many species. However, few studies have focused on the importance of environmental factors and resource availability in the success of wildlife reintroductions. The goal of this study was to see if location influences the short-term reintroduction success of captive-bred Texas horned lizards (Phrynosoma cornutum). Specifically, I monitored diets, growth rates, and survival of over 250 lizards reintroduced to 2 locations in Mason Mountain Wildlife Management Area (Mason County, TX) for 3 months. Diet, growth rates, and survival all differed between the two locations. The findings of this study suggest that environmental factors can play an important role in the reintroduction success of Texas horned lizards. Future research will focus on identifying specific habitat characteristics that may contribute to the observations of this study such as prey availability, vegetation, thermal habitat quality, and soil permeability.

Antibiotic resistance has been increasing rapidly; however, the amount of new and effective antibiotics is declining. One area of growing interest is the use of metal nanoparticles because they are relatively easy to make and can be synthesized into different shapes, sizes, and with various chemical properties. In particular, zinc oxide nanoparticles have shown to be effective against various bacterial strains; however, the mechanism that zinc oxide utilizes to exhibit its antimicrobial activity is still unknown. It is also not clear what properties of zinc oxide such as size or proximity to bacterial cells are critical for its antimicrobial activity. In order to gain a better understanding of the mechanism behind zinc oxide’s antimicrobial activity, we tested Staphylococcus aureus with various zinc oxide particles under different conditions. Specifically, we looked at whether particle size, contact with bacterial cells, and media type influenced antimicrobial activity. Our results suggest that particle size does not influence zinc oxide activity, but media type significantly impacts antimicrobial activity. Physical contact, although more effective, is not absolutely required to see inhibition of bacterial growth. Understanding the mechanisms that zinc oxide utilizes may guide design for future particles that will improve their effectiveness.

Alzheimer’s disease (AD) is a progressive neurodegenerative disease that is projected to affect almost 14 million American adults by the year 2050. While the prevalence of this detrimental disease is rapidly increasing in the United States, researchers have established the key pathologies connected to AD, including the development of extracellular, amyloid beta (Aβ) plaques, and intracellular, hyperphosphorylated, neurofibrillary tau tangles. Overall, AD engenders general atrophy of the brain and damage to key brain regions including the cerebral cortex and hippocampus, the main brain region responsible for the neural mechanisms of learning and memory. AD pathologies develop in these regions, which commonly results in neuronal death. The presence of AD pathologies, such as Aβ, activates microglial cells in the brain. Glial cells are the most common brain cells that provide support to neurons. Microglia specifically serve as resident immune cells in the brain, clearing cellular debris, such as dead neurons. Therefore, microglia play a key role in the progression of several neurodegenerative diseases. The activation of microglial cells results in an increased secretion of effector proteins, known as pro-inflammatory cytokines. These are released when inflammatory agents, such as Aβ, are present in the brain. Microglial cells commonly produce pro-inflammatory cytokines, such as TNF-α. Although microglial activation is advantageous at first, continual activation of microglial cells results in a constant inflammatory state. Chronic inflammation can lead to detrimental tissue damage that plays a vital role in neurodegeneration. Another key AD pathology, oxidative stress, is connected to chronic inflammation. Oxidative stress develops when the antioxidant system is unbalanced, resulting in the accumulation of reactive oxygen species (ROS). The presence of inflammatory agents and ROS have the potential to activate microglial cells. Accordingly, our lab utilizes microglial cells to study the harmful effects of inflammation on the brain. Dr. Kayla Green’s lab in the TCU Chemistry Department has successfully created compounds that act as potent antioxidants, L2 and L4. We collaborate with Dr. Green’s lab to research the possible rescue effects of L2 and L4 against inflammation in immortalized, BV2 microglial cells. In our lab’s previous research, we have demonstrated that both L2 and L4 have the capacity to rescue BV2 cells and increase cell survival during oxidative stress. Moreover, the main purpose of the current experiment is to further study the effects of these compounds against key AD pathologies, to understand their therapeutic potential against inflammation in vitro. In the current experiments, we utilized lipopolysaccharide (LPS), an element from the cell wall of gram-negative bacteria, to induce an inflammatory response in BV2 cells. First, we determined several timepoints and concentrations in which LPS treatment successfully induced the secretion of TNF-alpha. Next, we pre-treated cells with the compound, L4, for one hour prior to LPS treatment, to study the possible rescue effects of the drug against pro-inflammatory cytokine production. We are currently determining which concentration of L4 is the most therapeutic against pro-inflammatory cytokine production in BV2 cells.

Changes in thyroid hormones levels have been associated with alterations in somatic development and growth. However, recent studies have shown that alterations in thyroid hormone levels during early life stage (ELS) development can lead to long-term changes in reproduction. Specifically, fathead minnows that have been exposed to propylthiouracil (PTU) experienced a 50% reduction in fecundity. The purpose of this study was to determine if ELS thyroid disruption led to an alteration of reproductive behaviors in male fathead minnows. To accomplish this, larval fathead minnows were exposed to PTU and reproductive behaviors were quantified. Results showed that PTU-exposed fish demonstrated significantly fewer reproductive behaviors than those in the control group. This data provides an explanation for the previously observed 50% decrease in fecundity in the fathead minnows exposed to PTU and provides further evidence that ELS thyroid disruption can interfere with the display of key and ecologically-relevant behaviors later in life.

The rainbow trout, Oncorhynchus mykiss, is a partially migratory organism, that has been used recently to study the genetic control of migration. Much of this research has taken place at a unique site in Sashin Creek, Alaska, where the resident O. mykiss population is completely isolated from the migratory population. However, it is unknown the extent to which findings here are shared with other populations. Here we used a fine-scale genome-wide sequencing approach known as pooled sequencing to gather genetic data from 174 fish in two locations – Sashin Creek, Alaska and Little Sheep Creek, Oregon. Four sequenced pools were developed based on phenotype and population. We then measured differentiation between the populations to identify regions that may be correlated with the resident or migratory phenotype in both populations. We were able to locate 8 genes in 16 regions of shared elevated FST, and 17 genes over 16 regions with a significant Tajima’s D value that were specific to either the migratory or resident phenotype. These findings indicate specific genes and chromosomal regions that may be important in the regulation of migratory tendency in this species.

Mercury (Hg) is found in the environment in excess of historic baselines throughout the globe because of widespread atmospheric emissions of inorganic mercury (IHg) from anthropogenic sources such as coal-fired power plants and artisanal gold mines. In aquatic ecosystems, Ihg deposited from the atmosphere is converted by bacteria to methylmercury (MeHg), a bioavailable neurotoxin that adversely affects the health of vertebrates including humans and wildlife. Because IHg deposition varies across the landscape, it is necessary to monitor MeHg levels in aquatic food webs of individual waterbodies. This is a challenge because there are millions of river miles and lakes in the U.S. Shoreline spiders that feed on MeHg-contaminated emergent aquatic insects have been proposed as sentinel species to monitor MeHg contamination. Sentinel species are species which serve to map the bioavailable fraction of pollution in an ecosystem by retaining the pollutants in their tissue. The objective of this study was to test the hypothesis that shoreline spiders can be used as sentinels to evaluate MeHg contamination of river food webs. Our study focused on the Clear and West forks of the Trinity River. A pilot study in 2016 indicated the two forks have different levels of MeHg contamination. From June to August 2019, we collected over 1000 long-jawed orb weaver spiders (Tetragnathidae) along the shorelines of the two forks of the river. Spiders were preserved in 95% ethanol and sorted by leg length into different size categories. Mercury was analyzed using direct Hg analysis. Concentrations of Hg in spiders increased with spider size and was higher in the Clear Fork than the West Fork. A follow up study confirmed that fish in the Clear Fork had higher concentrations of MeHg than in the West Fork. This is one of the first studies to demonstrate that shoreline spiders can be used as sentinels of MeHg contamination in river ecosystems.

Across North America, bats are being killed in large numbers at wind energy facilities and there is concern that this level of mortality threatens bat populations. Currently three species of migratory tree bats, including two Lasiurus species, comprise ~75% of all known fatalities; however, as wind energy development expands into new areas (e.g., the southwestern U.S.) there is the potential for new species to be impacted. Ongoing work in our labs has indicated that that our current understanding of the distribution of Lasiurus species across North America is limited, at best, and that more species are impacted by wind energy development than previously thought. Accurate knowledge about which species are being impacted where, and to what extent, will greatly improve the implementation of effective mitigation strategies. We obtained 19 bat fecal samples from wild-caught Lasiurus bats from a study being conducted at Texas State University to improve the species-specific effectiveness of an ultrasonic acoustic deterrent (UAD) at deterring bats from approaching operational wind turbines. Based on morphology, these wild-caught bats were identified as eastern red bats (L. borealis), but it is possible that some of the individuals were western red bats (L. blossevillii). We extracted DNA from the bat fecal samples and amplified the COI mitochondrial gene to determine the correct species identification for each sample. The final sequencing reactions are underway, and the results will be available soon. These data will improve the accuracy of the results from the flight cage study at Texas State University and will contribute to improving strategies to reduce bat fatalities at wind energy facilities.