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.

Although wind energy facilities are a growing source of renewable, clean energy, they have been shown to contribute to increasing bat mortalities which could threaten the persistence of bat populations. This study aims to expand what we know about the biology and behavior of bat species impacted by wind energy development. Recent research has indicated that yellow bats (Lasiurus spp) are killed at wind energy facilities in the Rio Grande Valley of south Texas. We have limited understanding of the population biology or movement patterns in these species, so the extent to which wind turbine mortality may impact these bats is currently unknown. As part of ongoing research in our labs, I extracted DNA from 18 tissue samples collected from northern yellow bats (Lasiurus intermedius) at a wind energy facility in Willacy county, Texas in 2015. I amplified a region of the mtDNA, the COI locus, and will compare genetic diversity of these samples to a larger data set from wind energy facilities in nearby Starr and Hidalgo Counties that were studied in 2016 and 2017. Together, these datasets will improve our understanding of Lasiurus intermedius genetic diversity and population structure, and have the potential to provide much needed insights into the potential impacts of wind energy development on bats in southern North America.

The student hasn't submitted any abstract yet.

Mutations in BReast CAncer 1 protein (BRCA1) play a crucial role in DNA damage control such as double-strand DNA break repair mechanisms. Mutations in BRCA1 increase the chance of disrupted genetic integrity by its contributions to the development of breast cancer. BRCA1 must bind to its partner protein PABL2 (Partner and Localizer to BRCA2) in order to properly carry out its function in the repair mechanism pathway, but its conformation once bound to PALB2 is not clear. In its inactive state, PALB2 is known to remain in an alpha-helical coiled-coil homodimer conformation. Through this observation, we hypothesized that the intrinsically disordered region of BRCA1 on its binding surface will undergo a conformational change into an alpha-helical form. In order to test this hypothesis, we first created a truncated BRCA1, making it 50 amino acids long, then conducted nuclear magnetic resonance (NMR) experiments. Through the NMR experiments, we found that the binding interface of BRCA1 does change its conformation into a helical state, forming a coiled-coil heterodimer upon binding with PALB2.

The proper functioning of the protein PALB2 is vital to preventing tumor formation within breast tissues in individuals. Upon the detection of DNA damage, PALB2 and BRCA1 bind to each other along with BRCA2 to form a DNA repair complex. This complex then repairs DNA double-strand breaks in order to prevent the accumulation of DNA damage that leads to breast cancer. While both BRCA1 and BRCA2 have been extensively studied, a lot of information about the structure and function of PALB2 remains unknown. It is thought that BRCA1 and PALB2 bind via PALB2’s coiled-coil domain; however, how variants of unknown significance (VUS) affect this binding interaction is largely unknown. Further, while some of these VUS have been studied in vivo, cheaper and easier in vitro methods to measure their effect on binding affinity have yet to be formulated. Thus, we hypothesized that isothermal titration calorimetry (ITC) could be used as an in vitro testing method for assessing the effects of VUS within the coiled-coil domain of PALB2 on the binding event between PALB2 and BRCA1. Further, we hypothesized that a decrease in binding between the two proteins as measured by ITC would correlate with a decrease in DNA repair as measured in vivo. We tested the efficacy of this method by creating seven mutations within the coiled-coil domain of PALB2 and measuring the binding event of PALB2 to BRCA1 via ITC. Our results strongly suggest that the binding event is enthalpic in nature and can be adequately measured via ITC as evidenced by the correlation between our in vitro data and previous in vivo data.

Evaluating sex-specific differences in cellular immune function in a small fish model, the fathead minnow

Andrew Mielcuszny
Department of Biology

Advisor: Dr. Marlo Jeffries

Previous studies in the Jeffries lab have shown that male and female fathead minnows differ in their ability to fight and survive bacterial infections. Specifically, males have significantly higher rates of mortality upon infection than females. Despite this, few studies have sought to identify the sex-specific differences in specific immune processes that underlie the observed differences in survival following pathogen infection. The purpose of this study was to examine the sex-specific differences in phagocytic cell activity, a key innate immune response in which immune cells engulf and destroy pathogens. To evaluate phagocytic cell activity, kidney cells were isolated from male and female adult fathead minnows and their ability to phagocytose fluorescently-labeled E. coli was measured. The relative phagocytic cell activity of male and female fathead minnows will be presented in an effort to explain whether differences in phagocytosis contribute to differences in pathogen resistance.

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.

For this project, I mapped potential river otter habitat in the Dallas/Fort Worth metroplex based on literature data on known river otter habitat preferences in Texas. I will use this data along with GIS data on land use/cover/vegetation and distance from suitable water bodies, to determine where in the DFW river otters may prefer to live. This project is interesting and informative because in recent years more urban run-ins with river otters have been documented in the DFW. So knowing where they might like to be is good information for citizens to have, as river otters continue to make a comeback in numbers in the state of Texas.

Carnivorous plants inhabit nutrient-poor environments and supplement nutrient acquisition by capturing and digesting insect prey. Carnivorous adaptations have been hypothesized to be beneficial only in environments with high water and light availability. We hypothesized that plant morphology would change in response to resource availability, exhibiting traits that increase carnivory when light is abundant and exhibiting traits that increase photosynthesis when light is limited. In a field manipulation in Leon County, Texas, we examined the effects of feeding, shading, and their interaction on the morphology of the pitcher plant, Sarracenia alata. We employed a two-factor, cross-classified design, with shading (two levels, shaded and unshaded) and prey capture (two levels, fed and unfed) as factors. Eighty plants were haphazardly assigned to one of four treatments: (1) unshaded and fed (control); (2) shaded and fed; (3) unshaded and unfed; and (4) shaded and unfed. When light availability was reduced, plants produced pitchers that had smaller diameters, which is reflective of a photosynthetic morphology. Unfed plants exhibited reduced growth (produced fewer pitchers and had lower sum of pitcher heights). There was a significant interaction effect on estimated seasonal aboveground biomass: shading had no effect on the mass of unfed plants, but shading reduced the mass of fed plants. As the season progressed, competing vegetation reduced light availability to all pitchers. Plants in all treatments began to produce pitchers that were blade-like with a small, non-functional opening and a widened keel. This morphology would maximize light capture at the expense of prey capture. This experiment provides support for a theoretical model that suggests that carnivorous traits are only beneficial under conditions of high light availability. It also emphasizes the importance of periodic burns of carnivorous plant bogs to remove vegetation, thereby reducing light competition.

Bacillus anthracis is a bacterium that causes the deadly disease anthrax and has been used in bioterrorism. We are looking to investigate what genes within the chromosomal DNA contribute to the virulence of Bacillus anthracis. In this study, we screened a transposon library of B. anthracis ‘knock-out’ mutants for susceptibility to reactive oxygen species used by the immune system. A broad in vitro hydrogen peroxide screen was performed on 1,953 transposon mutants, and after several rounds of in vitro screening, 40 mutants were identified as consistently attenuated in the presence of hydrogen peroxide. Four of these mutants were then tested in the invertebrate model, Galleria mellonella, to assess virulence in an animal model. Mutants with phenotypes that repeated in both assays were prioritized for characterization. The location of the transposon insertion in one of the mutants was successfully identified. Identifying these novel genes contributing to the bacterium’s virulence will provide a better understanding of B. anthracis pathogenesis and may provide potential targets for combatting anthrax.

The bacterium Bacillus anthracis, the causative agent for the disease anthrax, possesses two plasmids that contribute significantly to virulence. Besides plasmids, certain chromosomal genes also contribute. In previous studies, our lab discovered that the chromosomally encoded ClpX gene is essential for virulence in B. anthracis. ClpX is an ATPase that is part of the ClpXP proteasome found in many bacteria. Loss of ClpX in B. anthracis Sterne results in increased susceptibility to cell wall targeting antibiotics like penicillin and daptomycin. However, the mechanism behind ClpX’s role in antibiotic resistance is not understood as it is likely that multiple pathways are affected by the loss of this global protease. We recently conducted a microarray to find which genes are up or down regulated in ClpX compared to wild-type (WT) B. anthracis. 119 genes had disrupted regulation and several of these had been connected to cell-wall active antibiotics like penicillin. In this study, we focused on three of these genes: MsrA, GlpF, and SigM. We confirmed the microarray results and showed that MsrA, GlpF, and SigM gene expression in our ClpX strains significantly differs from the wild-type B. anthracis Sterne via QPCR. Insertional knockout mutants were made for GlpF and SigM to test whether these genes were necessary for antibiotic resistance. We are currently testing these mutants in penicillin and daptomycin to assess their phenotypes. We found that loss of SigM results in increased susceptibility to penicillin and are currently studying the effect of daptomycin on SigM and GlpF. We will test the virulence of both mutants in our invertebrate animal model G. mellonella. This will hopefully provide better understanding on the mechanism behind ClpX’s antibiotic resistance.

Studies have shown that males and females differ with regard to their ability to survive pathogen infections. The fathead minnow is a newly developed model for immunotoxicity; however, few studies have compared male and female immune responses following pathogen exposure. The purpose of this study was to examine sex-based differences in pathogen resistance and immune responses following exposure to a pathogen in adult fathead minnows (Pimephales promelas). To accomplish this, fish were bacterially infected with Yersinia ruckeri and the immune system’s ability to respond was monitored. Additionally, genes that are known to be expressed during the immune response initiation were measured quantitatively, providing insight into the molecular effect in minnows. At the whole organism level, male fish were less able to survive pathogen infection relative to female fish. At the tissue level, both male and female pathogen-injected fish had decreased hematocrit percentages compared to the fish injected with a saline solution, but did not differ from each other. At the molecular level, increased gene expression of interleukin 1β was seen in pathogen-injected males compared to pathogen-injected females and both sham-injected sexes, indicating that pathogen-injected males mounted a larger inflammatory response at the molecular level. Taken together, this evidence suggests that the increased mortality observed among males earlier in the exposure to the pathogen may be due to the upregulated inflammatory response rather than the effects of the pathogen itself.

Pollen transfer among flowers contributes to genetic diversity and the maintenance of plant populations through the production of seeds. Decreased pollen receipt can result in fewer offspring. This is known as pollen limitation. We conducted field and laboratory experiments in a population of Sarracenia alata in Leon County, Texas in 2019 to examine 1) the effect of floral herbivory by the pitcher plant moth, Exyra semicrocea, on pollen availability and 2) the impact of pollen receipt on seed quantity and 3) seed quality. We found that floral herbivory significantly decreased the number and mass of anthers in flowers, and that a high pollen load significantly increased the number of seeds produced compared to low-pollen and control flowers. We found no differences in offspring quality among different pollen treatments based on germination traits. Pollen limitation occurs in S. alata and may pose a conservation risk when paired with other ecological disturbances.

Bacillus anthracis is a gram-positive, spore-forming bacterium and the causative agent of the deadly disease anthrax. The B. anthracis genome consists of chromosomal genes and the pXO1 and pXO2 plasmids that strongly contribute to the bacteria’s deadly nature. While the virulence factors associated with the plasmids have been extensively studied, we believe there are still undiscovered chromosomal genes that may also have important virulence factors. To identify novel chromosomal genes associated with B. anthracis virulence, we screened a transposon mutant library of B. anthracis Sterne strain for increased sensitivity to reactive oxygen species. Reactive oxygen species, such as hydrogen peroxide, have many functions in mammalian immune defenses and wild type B. anthracis is able to subvert this host defense. Sensitivity to reactive oxygen species was tested through in vitro hydrogen peroxide assays and after several rounds of screening, eight mutants were confirmed as susceptible. We next tested whether any of these mutants were attenuated in vivo using our invertebrate animal model, Galleria mellonella and found several mutants with decreased virulence. We are currently working on determining the location of the transposon insertion to find which chromosomal gene is disrupted. This could lead to the discovery of novel B. anthracis virulence genes and eventually possible treatment targets for future anthrax outbreaks and attacks.