Doctor of Philosophy / Department of Division of Biology / John Blair / The feasibility of restoration, which traditionally targets historical conditions, is questionable in the context of global change. To address this, my dissertation investigated (Chapter 2) the patterns of restoration establishment along a chronosequence of restored prairies with respect to nearby remnant prairies, (Chapters 3-4) responses of plant communities in restorations initiated using different methods (levels of species richness and sowing density) to drought, which is projected to increase in frequency, and (Chapters 5-6) the effects of propagule source and variation (mixing among sources) on restoration establishment and the generality of restoration outcomes across variable environments using reciprocal common gardens of multi-species restoration seedings. Chapter 2, published in Restoration Ecology, showed that restoration led to the recovery of desirable characteristics within several years, but restorations utilizing primarily fall-collected seeds likely diminished the representation of early phenology species, so biodiversity may be further enhanced by including early phenology species in seeding mixes. Chapters 3 and 4, published respectively in Ecological Applications and Applied Vegetation Science, examined the establishment of native plant communities after seeding and their responses to experimentally imposed drought. Both high seed mixture richness and high density seeding resulted in greater establishment of native, seeded species compared to low richness and low density treatments, and exotic species were less prevalent in high richness and high density treatments. However, we found little evidence of differential drought resistance, recovery, and resilience among treatments. This result coupled with increases in exotic species following drought suggest that other forms of active management may be needed to produce restored plant communities that are robust to climate change. Chapter 5 (published in Ecosphere) iii and Chapter 6 found that seed source affects individual species establishment, community structure, and productivity. However, there was no consistent advantage for any source, including local sources, across sites or species. This suggests that source effects on single species or effects observed at single locations should not be broadly generalized. Together, this dissertation shows that restoration can recover many characteristics of native prairies and that manipulation of seeding practices (seed mixture richness, seeding density, seed source) influence grassland establishment in terms of productivity, community structure, invasion, and the abundance and survival of individual species.

Master of Arts / Department of Geography / Kendra K. McLauchlan / Reconstructing fire regimes and fuel characteristics is an important aspect of understanding past forest ecosystem processes. Fuel sources and disturbance regimes throughout the upper Midwestern United States have been shown to be sensitive to regional climatic variability such as drought periods on millennial timescales. Yet, records documenting the complex connections between disturbance activity and the corresponding fuel source fluctuations in mesic deciduous forests and oak savanna forests in this region are limited. Thus, it has been difficult to provide a framework to evaluate drought conditions on fire activity and the relationships with fuel source fluctuations in this region. Here, I conducted high-resolution charcoal analyses of lake sediments from four sites in southeastern-southcentral Wisconsin (USA) to characterize fire activity and fuel source fluctuation in mesic deciduous forests and prairie-oak savanna over the last 10,000 years. I found that fire regimes across the four study sites have been asynchronous throughout the Holocene, due to site-specific differences that have strongly influenced local fire regimes. I also found that during periods of high fire activity the primary fuels were from arboreal sources, and during periods of low fire activity the primary fuels were from non-arboreal sources. However, fluctuations in fuel sources did not always correspond to changes in vegetation, or changes in fire frequency.

Master of Science / Division of Biology / Kristin Michel / To date malaria is the most important tropical disease, which is caused by Plasmodium sp. and vectored by anopheline mosquitoes. The mosquito’s immune system is one of the limiting factors of malaria transmission. Immune reactions, such as the prophenoloxidase (PPO) pathway result in the melanization of pathogens, and are effective at limiting parasite numbers. Novel strategies for malaria control aim to exploit the immune system to interrupt parasite transmission by boosting the immune responses in the mosquito vector. Serpins play a crucial role in regulating protease cascades involved in immunity of arthropods. In Anopheles gambiae, the major malaria vector in Sub-Saharan Africa, 18 SRPN genes encoding 23 distinct proteins have been identified. So far, two are identified as active inhibitors, and both affect parasite survival. This research aims to identify additional inhibitory serpins in An. gambiae and elucidate their potential function. Identification of such serpins will enhance our understanding of the immune system of this important vector species and may identify immunoregulators to be used in malaria control. SRPN7, 9, and 18 were tested for their ability to inhibit commercial proteases in vitro. Recombinant SRPN18 had no inhibitory activity, while SRPN7 and 9 inhibited several serine proteases. SRPN7, 9 and 18 were tested against two recombinant An. gambiae clip serine proteases (CLIPBs) that are required for activation of phenoloxidase and thus regulate melanization. Only SRPN9 strongly inhibited CLIPB9 in vitro, suggesting that this serpin is a potential negative regulator of melanization. This hypothesis is further supported by the finding that SRPN9 can inhibit PO activity in insect hemolymph, ex vivo. Taken together, this research identifies SRPN18 as the first non-inhibitory serpin described in mosquitoes. Additionally, this study describes the larval-specific SRPN7 as a functional inhibitor. Future studies on these proteins will elucidate their precise physiological functions. Finally, this thesis provides strong evidence that SRPN9 is a negative regulator of melanization in An. gambiae and may therefore affect pathogen survival within this important vector species.

Doctor of Philosophy / Division of Biology / Sherry D. Fleming / Ischemia/reperfusion (IR) events result in severe tissue damage and often death. The complex network of molecular and cellular mechanisms that contributes to intestinal IR-induced pathology has hindered a comprehensive understanding of IR-induced injury and limited the success of medical intervention. Although several of the mechanisms contributing to intestinal IR-induced injury have been identified, the initiating event(s) remains unclear. Mouse models have been instrumental in the unraveling of the many components and interactions that ultimately result in tissue damage. It is clear that leukocyte infiltration, complement activation, eicosanoid and pro-inflammatory cytokine production are involved. Toll-like receptors and antibodies also play critical roles. Based on the literature, and especially data demonstrating a significant role for anti-phospholipid antibodies, we hypothesized that ischemia induces phospholipid alterations that result in the exposure of a neoantigen which is recognized by anti-phospholipid antibodies. Furthermore, we hypothesized that endothelial cells are the primary cell type involved in the initial molecular events that result in intestinal IR-induced pathology. A mouse model of intestinal IR as well as an in vitro cell culture system was used to explore these hypotheses. Mass spectrometry-based lipidomics was utilized to assess lipid responses to IR and hypoxia/re-oxygenation (HR). No inherent differences in intestinal phospholipid composition were found between wildtype and several strains of knock-out mice. It was determined that the lack of antibody production by Rag-1[superscript]-[superscript]/[superscript]- mice is responsible for protection against intestinal IR-induced injury, as antibody is needed to induce prostaglandin E[subscript]2 production, through up-regulation of cyclooxygenase 2 transcription. Unexpectedly, the presence or absence of toll-like receptor 9 was found to be inconsequential for tissue damage caused by intestinal IR. The results of several analyses point to endothelial cells as being directly involved in IR-induced pathology. Importantly, the activation of phospholipid scramblase 1 has been identified as a potential molecular mechanism by which subsequent molecular and cellular responses are elicited as a consequence of IR.

Master of Science / Department of Entomology / Marcelo Ramalho-Ortigao / Sand flies (Diptera:Psychodidae) are vectors of parasites if the genus Leishmania, the causative agent of leishmaniasis, a neglected tropical disease in several countries around the world. Sand flies transmit Leishmania to suitable vertebrates during the blood meal and following a complex development parasites undergo within the fly. Many aspects of the Leishmania development within the sand fly vector are well known, however details about how sand fly molecules affect the parasite are still not yet known. Our group previously identified that RNAi knockdown of PpChit1, a midgut specific chitinase from the sand fly Phlebotomus papatasi, led to a significant decrease in the load of Le. major. In this study, we assessed potential fitness effects of antisera anti-PpChit1 on three laboratory-reared sand fly species (P. papatasi, Phlebotomus duboscqi, and Lutzomyia longipalpis). Our results suggest that feeding sand flies with anti-PpChit1 sera led to a one day delay in the onset of oviposition, and also suggested that anti-PpChit1-fed flies survived on average up to three days longer that control flies. Analyses of the peritrophic matrix (PM) indicated a significant increase in thickness 72 hours post anti-PpChit1 feeding compared to control sera. Altogether the results suggest that feeding sand flies with anti-PpChit1 likely affects the kinetics of sand PM, which in turn affects the flow of nutrients and certain aspects of sand fly fitness. In the course of this study, we also evaluated the ability of American Foxhounds naturally infected with Leishmania infantum to transmit these parasites via bites of phlebotomine sand flies to suitable vertebrates. Since 1999, an outbreak of canine visceral leishmaniasis (CVL) has been reported in the U.S especially among Foxhounds. The ability of sand flies to pick up and transmit this pathogen represents an important health risk for companion dogs and humans. Our results indicate that Foxhounds naturally infected with Le. infantum are highly infectious to sand flies and that the parasites are able to fully develop within these vectors and de successfully transmitted during blood feeding. Thus, the risk exists for these parasites to become endemic in North America where sand flies are also known to occur.

Doctor of Philosophy / Division of Biology / Walter K. Dodds / Tallgrass prairie evolved with fire-grazer interactions. Fire and grazing are vital processes for maintaining grasslands and cattle production, and therefore will be continued as land management schemes. The effects of fire and grazers on prairie streams are understudied, but may significantly influence stream ecology. This dissertation examined how prescribed burning, bison grazing, and patch-burn grazing (by cattle) influence water quality, stream biota, and riparian amphibians and reptiles at Konza Prairie, Kansas, or Osage Prairie, Missouri. Using Global Positioning System, we monitored bison and cattle distribution throughout watersheds. The immediate effects of prescribed burning were examined at both Konza and Osage Prairies. The impacts of bison on water quality were determined by using a long-term dataset from Konza Prairie and compared watersheds with and without bison. Amphibian and reptile assemblages were monitored for two years at Osage, and assemblage data were analyzed using redundancy analysis, permuted analysis of variance, and occupancy modeling. A patch-burn grazing experiment occurred for 5 years at Osage (2 years pretreatment data and 3 years of treatments) and was analyzed using a before-after, control-impact design. Prescribed burning had minimal effects on water chemistry. At Konza Prairie, bison did not alter water quality likely because they spent negligible time (<5%) in streams. Contrarily, cattle at Osage Prairie significantly increased stream concentrations of total suspended solids, nutrients, Escherichia coli bacteria, algal biomass, and primary production. Unlike bison, cattle spent significant time (~21%) in streams if allowed access to riparian zones. In watersheds with cattle excluded from streams by riparian fencing, water quality contaminant concentrations increased significantly, but not to the magnitude of unfenced streams. Amphibian abundance and richness were not different among patch types; instead, they were restricted to specific basins. However, reptiles displayed preference for certain patch-types, and had the highest abundance and richness in watersheds with fire and grazing. These results have implications for natural resource management. Riparian fencing of cattle may be a useful practice in areas where water resource protection is the priority. However, overland flow may alter water quality in watersheds with grazers despite fencing. Land managers will need to define management objectives and accept trade-offs in water quality, amphibian and reptile habitat, and cattle production.

Master of Science / Division of Biology / Revathi Govind / Toxigenic Clostridium sordellii causes uncommon but highly lethal infections in humans and animals. Recently, an increased incidence of C. sordellii infections has been reported in women undergoing obstetric interventions. Pathogenic strains of C. sordellii produce numerous virulence factors, including sordellilysin, phospholipase, neuraminidase, and two large clostridial glucosylating toxins, TcsL and TcsH. Recent studies have demonstrated that TcsL toxin is an essential virulence factor for the pathogenicity of C. sordellii. In this study, we identified and characterized TcsR as the toxin gene (tcsL) regulator in C. sordellii. High-throughput sequencing of two C. sordellii strains revealed that tcsR lies within a genomic region that encodes TcsL, TcsH, and TcsE, a putative holin. By using ClosTron technology, we inactivated the tcsR gene in strain ATCC 9714. Toxin production and tcsL transcription were decreased in the tcsR mutant strain. However, the complemented tcsR mutant produced large amounts of toxins, similar to the parental strain. Expression of the Clostridium difficile toxin gene regulator tcdR also restored toxin production to the C. sordellii tcsR mutant, showing that these sigma factors are functionally interchangeable.

Doctor of Philosophy / Division of Biology / Theodore J. Morgan / Thermal stress impacts animals around the globe and understanding how organisms adapt to changes in temperature is of particular interest under current climate change predictions. My research focuses on the evolutionary genetics involved in cold tolerance and plasticity of cold tolerance using both artificially selected and naturally segregating populations, while tying the genes of interest to their physiological components. First I address cross-tolerance of stress traits following artificial selection to a non-lethal cold tolerance metric, chill-coma recovery. Using these artificial selection populations, we found that stress traits such as desiccation tolerance, starvation tolerance, acclimation, and chronic and acute cold tolerance do not correlate with level of cold tolerance as defined by chill-coma recovery time. We next assessed lifetime fitness of these different cold tolerance lines and found that only at low temperatures did fitness differ among cold tolerance levels. We then analyzed gene expression differences between resistant and susceptible populations at three time points to understand where selection pressures are hypothesized to act on genomic variation. Our gene expression analyses found many differences between resistant and susceptible lines, primarily manifesting themselves in the recovery period following cold exposure. We next utilized a community resource, the Drosophila melanogaster reference panel, to identify naturally segregating variation in genes associated with cold acclimation and fitness. We specifically asked if long- and short-term acclimation ability had overlapping genetic regions and if plasticity values from constant rearing environments were associated with demographic parameters in fluctuating environments. We found that long- and short-term acclimation are under unique genetic control and functionally tested several genes for acclimation ability. We also found that acclimation ability in constant environments and fitness in fluctuating environments do not correlate, but that genotypes are constrained in their fitness abilities between a warm and cool environment. Our analyses describe several novel genes associated with cold tolerance selection and long- and short-term acclimation expanding our knowledge of the complex relationship between demographic components and survivorship as well as a unique investigation of the change in gene expression during cold exposure.

Master of Science / Department of Biology / Brett K. Sandercock / The migratory period is a critical time for birds. Population declines in long-distance migratory birds have been associated with trophic mismatches among climate change, timing of food availability, and timing of migratory movements. Studies on migratory songbirds have been limited to eastern North America and Europe, and migration strategies of birds may differ along the Pacific flyway. We evaluated the stopover ecology of five species of migratory songbirds at a coastal site in northern California. We found variation in changes in timing of spring migration, but consistent changes in timing of autumn migration over a 22-year period. Timing of spring migration advanced for Swainson’s Thrushes (Catharus ustulatus) and Yellow Warblers (Setophaga petechia), was compressed for Pacific-slope Flycatchers (Empidonax difficilis) and Wilson’s Warblers (Cardellina pusilla), but was protracted for Orange-crowned Warblers (Oreothlypis celata). In contrast, timing of autumn migration was delayed for Pacific-slope Flycatchers, Orange-crowned Warblers, and Yellow Warblers, but was protracted for Wilson’s Warblers. Warm, wet conditions were associated with advanced spring migration, and warm, dry conditions were associated with delayed autumn migration. Changes in timing of migration related to climate conditions were strongest for Pacific-slope Flycatchers and Orange-crowned Warblers. Stopover duration of our five study species was longer than songbirds in eastern flyways, and on average, was longer in spring than autumn. Pacific-slope Flycatchers and Yellow Warblers had longer stopovers in spring, whereas Swainson’s Thrushes and Wilson’s Warblers stopped had longer stopovers in autumn. Birds captured at low body mass had longer stopovers in spring and autumn. Migratory birds in western North America may have different migration strategies because of differences in climate and geography, but more study is needed to discover migratory routes and compare our results to other stopover sites along the Pacific flyway. Understanding differences in migration strategies of different populations of the same species are important in directing conservation efforts, especially in light of ongoing climate change.

Doctor of Philosophy / Department of Diagnostic Medicine/Pathobiology / Sanjeev Narayanan / Antimicrobial resistance (AMR) has arisen as one of the most important public health concerns in the last 60 years. AMR results from pathogenic strains of bacteria adapting to antimicrobial-containing environments through mutations or through horizontal gene transfer (HGT) of genetic material containing resistance genes. Conjugation machinery offers an efficient method for acquisition of AMR and virulence genes, which may be responsible for propelling the evolution of pathogenic bacteria. This dissertation explores the factors, specifically catecholamines and antimicrobials that influence the conjugation frequencies of enteric bacteria including Salmonella, E. coli and Enterococcus. We found that the catecholamine norepinephrine (NE) at physiological concentrations enhanced conjugation efficiencies of a conjugative plasmid from a clinical strain of Salmonella Typhimurium to an E. coli recipient in vitro. Additional experiments determined the influence of the antimicrobial concentrations above, equal to and below the minimum inhibitory concentration (MIC) under in vitro conditions on conjugation efficiencies using an Enterococcus to Enterococcus mating pair in addition to the Salmonella to E. coli mating pair. Conjugation occurred in all concentrations, but efficiencies of transfer were consistently low in 0 MIC and 1 MIC, with increased activity both above and below 1 MIC. These data were fit to a previously described mathematical model and the rate constant E that relates the rate of gene transfer to drug concentration was determined. The data showed highly similar patterns of conjugation efficiencies when compared to the rate constant E. A final study we measured conjugation frequencies when donor Salmonella Typhimurium and the E. coli recipient were exposed to both variable concentrations of oxytetracycline and NE. Conjugation was increased pre- and post- MIC, but conjugation frequencies were not enhanced further by the combination of the oxytetracycline and the NE. This dissertation defines the role of outside factors in conjugative gene transfer, and may provide future insight into better control of AMR.