Investigating reproductive success and endocrine regulation of mating strategies in male medaka

by Lauren Closs

Faculty mentor: Dr. Dianne Baker

Mate guarding, when two males compete for one female, is a reproductive strategy seen across a variety of vertebrate species. This often leads to hierarchical relationships, in which one male exerts dominance over other, subordinate males. However, the physiological mechanisms that promote dominance or subordinance in males remain largely unexplored. This study investigates the reproductive success and endocrine signals of these reproductive strategies in Japanese medaka (Oryzias latipes). To identify dominant and subordinate males, triads consisting of two males of different genotypes and one female were observed repeatedly for 5 days. Male reproductive success was determined by genotyping embryos from each female. We found that the number of eggs fertilized by dominants and subordinates did not differ (p=0.29), indicating that dominant behavior does not guarantee reproductive success and that subordinate males may successfully fertilize eggs using sneaker male tactics. We hypothesized that these behaviors are linked to activity in the reproductive endocrine axis. To test this hypothesis, we quantified pituitary luteinizing hormone (LH) and follicle stimulating hormone (FSH) in dominant and subordinate males using ELISAs. While FSH did not differ between the groups, LH was unexpectedly higher in subordinate males (p=0.047). This indicates that either LH production is stimulated, or its pituitary release is inhibited in subordinates. To investigate these opposing explanations, we measured mRNA levels of LH, FSH, and GnRH receptors in the pituitary, and GnRH and AVT in the brain of dominant and subordinate males using qPCR. Mean differences between dominants and subordinates were not significant for any gene. Dominant fish expressed higher lhb in 8/12 tanks, indicating that LH production is not stimulated in subordinates, but as the transcripts for GnRH and its receptors also did not differ, further studies are needed to determine the mechanism by which LH release may be inhibited.

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Assessing the presence and concentrations of microplastics in the gizzards of Virginia waterfowl

by Thomas Bustamante

Faculty Advisor: Dr. Andrew Dolby

Microplastics are defined as plastic fragments that are smaller than 5mm. These particles have become a ubiquitous water pollutant in recent years. While a substantial amount of research on their impacts on marine ecosystems has been conducted, the effect of microplastics on freshwater food webs remains poorly understood. In this study, we assessed the presence and concentrations of microplastic particles in the gizzards of the Canada Goose (Branta canadensis), Ring-necked Duck (Aythya collaris), Long-tailed Duck (Clangula hyemalis), Bufflehead Duck (Bucephala albeola), and Mallard (Anas platyrhynchos) hunted in the Piedmont and Coastal Plain of Virginia. Gizzards were bisected and had their contents removed for analysis. Collected gizzard contents underwent density separation in order to separate plastics from the sediment within gizzards. Isolated particles were then visually inspected under a dissecting microscope. Preliminary results show that with contamination taken into account, 90% of birds sampled contain microplastic fibers in their gizzards. These range in concentration from about 0.08-11.49 fibers/gram of gizzard material. All plastics were secondary fibers, meaning they originated from larger plastics that broke down over time. This provides further evidence that waterfowl not only ingest microplastics, but retain them in their digestive systems. Infrared spectroscopy was done on one fiber; it was determined to be polyethylene. As we move forward, we plan to incorporate chemical digestion and a more efficient chemical analysis in our methods. Further studies will also examine microplastic spatial distributions, as well as microplastics in the intestinal lining and lumen of these birds. This work will be able to help us understand the threat microplastics pose to organisms and ecosystems, as well as help us understand their geographic distributions.

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Carbon Sequestration and Storage Estimates of Landscaped Trees on the University of Mary Washington Campus

by Daniel Milliken

Faculty Mentor: Dr. Alan Griffith

Trees play an important ecological role within the urban environment. They improve public health, provide environmental support and provide aesthetic benefits to cities. To help provide a better understanding on the services that our trees provide, researchers have come up with a way to place economic (monetary) value on these services they provide. Our study will explore the ideas of ecosystem values and answer the following questions: What are the services that the trees located on the University of Mary Washington (UMW) campus provide for the faculty and student population? Specifically, what amount of carbon is removed from the atmosphere by each species of tree through sequestration and storage? This independent research project used the United States Department of Agriculture Forest Service’s software suit, i-Tree Eco, to quantify the ecosystem benefits that the University of Mary Washington’s urban forest conveys to its community.

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Effects of Probiotics on Inflammatory Responses in Neuronal Tissue

by Laura O’Dea, Meredith LeBel, Nicole Haynes

Faculty mentor: Dr. Deborah O’Dell

Alzheimer’s disease (AD) is a neurodegenerative disease that affects more than 40 million people. While the pathophysiology has yet to be fully elucidated, some studies suggest AD associated chronic inflammation is caused by hyperactive microglia that produce pro-inflammatory factors. Probiotics have been shown to have anti-inflammatory properties and may influence neurochemistry via the gut-brain-axis, which controls communication between the intestines and brain, crossing over the blood brain barrier (BBB). A model of the BBB was constructed with a double transwell system to clarify the effects of probiotics on cerebral inflammation. Microglia cells grown in the basolateral chamber were co-cultured with endothelial cells in the upper compartment while an astrocyte monolayer separated the two compartments. Once the system was exposed to human peripheral blood T-cells and combined with histamine (probiotic anti-inflammatory product), formic acid (probiotic inflammatory product), both, or neither, the microglial medium was collected and analyzed for tumor necrosis factor α (TNFα) and interleukin-10 using ELISA. ANOVA and T-Tests were run and showed no significant results, except for the histamine and formic acid combination. In the combination treatment, levels of TNFα were slightly different than the control (p = 0.00006), contrary to what was expected. Under these conditions, probiotics do not reduce inflammation in the brain and thus cannot effectively treat AD patients. However, in the future, more experiments should be conducted with multiple inflammatory and anti-inflammatory molecules as there could be overlapping interactions between several probiotic products that produce advantageous metabolic effects and mitigate elevations in inflammatory responses.

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Embryonic development of the stress hormone axis in two model teleost species

by Rachel Summers

Faculty mentor: Dr. Dianne Baker

Glucocorticoid hormones mediate stress responses in all vertebrates, from teleost fishes to mammals. In adult teleosts, the primary glucocorticoid, cortisol, is synthesized within interrenal tissue via enzyme-mediated reactions regulated by the hypothalamic-pituitary-interrenal (HPI) axis in response to stressors. The hypothalamic peptide corticotropin-releasing hormone (CRH) stimulates release of the pituitary protein adrenocorticotropin hormone (ACTH), which stimulates cortisol production in interrenal cells. Cortisol affects target cells via two types of receptors, the glucocorticoid receptor (GR) and mineralocorticoid receptor (MR). The timing and sequence of events leading to a fully functioning HPI axis in developing nonmammalian vertebrates is not fully known. Addressing this gap, we measured expression of genes involved in cortisol synthesis and signaling throughout embryogenesis in two teleosts, the zebrafish (Danio rerio) and Japanese medaka (Oryzias latipes). We isolated RNA from pools of embryos collected at multiple developmental stages and synthesized complementary DNA (cDNA) by reverse transcription. Using cDNA as a template, we measured relative expression of key HPI genes, including CRH, melanocortin type 2 receptor (MC2R), steroidogenic acute regulatory protein (StAR), 11 β hydroxysteroid dehydrogenase (HSD2), and MR by quantitative polymerase chain reaction (qPCR). We measured cortisol throughout embryogenesis using an enzyme-linked immunosorbent assay (ELISA). We found noteworthy differences in expression profiles for all measured genes between species. Notably, we saw large differences in magnitude for changes in CRH, MC2R, and StAR expression. HSD2 showed different patterns of expression in zebrafish and medaka. Temporal cortisol patterns differed between species. Medaka cortisol increased significantly for hatch, whereas zebrafish saw a significant decrease.