Master of Science / Department of Biology / Jack Cully, Jr. / Disturbance is defined as any discrete event that disrupts ecosystem, community, or population structure and changes resources, substrate availability, or the physical environment. Habitat use by an organism is based on its perception of where to maximize its own fitness, and can be altered in response to disturbance-induced changes in resources, substrate, or physical features modified by disturbance. Disturbance-induced changes to vegetation structure reshape a small mammal’s surrounding physical environment and/or resources, and may influence its utilization of an area. Effective wildlife and resource management is dependent on a thorough understanding of how individual species and communities utilize their surroundings and how disturbance affects a species’ response to changes in its surroundings. We investigated seasonal habitat associations of three small mammal species and for overall species diversity across a gradient of military combat-vehicle disturbance intensities at the Fort Riley Military Reservation, Kansas. Deer mouse (Peromyscus maniculatus) abundance did not vary across a categorical gradient of disturbance created by military-combat vehicles, regardless of season. Western harvest mouse (Reithrodontomys megalotis) abundance was associated with more highly disturbed areas irrespective of season. Prairie vole (Microtus ochrogaster) abundance was associated with habitat that was less disturbed in the spring but more highly disturbed in the fall. Shannon diversity of the small mammal community was higher in the more highly disturbed areas regardless of season. This research shows that small mammals respond to disturbances created by military training with combat vehicles in a species-specific manner, and indicates that there may be differences in the effects of military training versus natural or agricultural disturbances on the abundance and diversity of small mammals. This is an important consideration given that the Department of Defense manages more than 12 million ha of land in the United States, and is charged under the Sikes Act with conserving natural resources on these lands, including biological diversity. Thus, the findings of other ecological research on the effects of disturbance on small mammals may not be directly applicable to the types of disturbances that occur on military lands, which underscores the need for further research on the specific effects of military-training activities on species’ responses.

Doctor of Philosophy / Department of Biology / Walter K. Dodds / Much of the North American tallgrass prairie ecosystem has been converted to cropland or urbanized. One threat to the remaining prairie ecosystems, and the streams within, is woody vegetation encroachment. Stream productivity, measured as metabolism, is a fundamental process comprised of gross primary production (GPP) and (CR) community respiration. Understanding GPP and CR is important because these processes are vital to ecosystem function and can be impacted by a change in canopy cover. First, I investigated improvements in existing methods for estimating whole-stream metabolism as estimated from diel patterns of oxygen (O2). I compared measured and modeled O2 and aeration (a physical parameter required for measurement of metabolism) rates to determine if direct measurement of aeration is necessary and the importance of temperature correction of metabolism. Modeling was moderately successful in determining aeration rates, and temperature correction of GPP and CR substantially improved model fits. Second, effects of woody vegetation encroachment on prairie stream function were investigated. Stream metabolism was measured for four years in duplicate reaches with varying canopy cover (closed canopy, naturally open canopy, and vegetation removal reaches). The removal reaches had closed canopy for the first two years and open canopy for the last two years. Canopy cover increased CR rates and had minimal effects on GPP. Third, the same experiment was used to determine the effects of woody vegetation encroachment on prairie stream ecosystem structure and food web interactions. Chlorophyll a and filamentous algal biomass were greater in naturally open and vegetation removal reaches, although the effects were stronger on filamentous algal biomass. As canopy cover decreased, the filamentous algal biomass to chlorophyll ratio increased, indicating a shift in algal community structure. Stable isotope analysis indicated some shift in pathways of nitrogen and carbon flux into the food web related to degree of canopy cover, but overlap in the signature of food sources made distinct food sources difficult to identify. The data indicate that riparian encroachment can influence ecosystem structure and function in prairie streams and restoration to remove woody riparian cover may restore some ecosystem features of naturally open canopy streams.

Doctor of Philosophy / Division of Biology / Jeroen Roelofs / The ubiquitin-proteasome pathway is the major pathway of selective protein degradation in the cell. Disruption of this pathway affects cellular protein homeostasis and contributes to diseases like cancer, and neurodegeneration. The end point of this pathway is the proteasome, a complex protease formed by 66 polypeptides. Structurally, it can be subdivided into the Core Particle (CP) and the Regulatory Particle (RP). The CP harbors the proteolytic sites, whereas, the RP contains six orthologous AAA-ATPases, the Rpt proteins. These Rpt’s are essential for proteasome function and are at the interface between RP and CP. The work in this thesis focuses on the Rpt subunit Rpt5 from yeast. The C-terminal tail of Rpt5 has been shown to contribute to the binding with the CP. However, our study showed it is also essential for the interaction with Nas2, one of nine proteasome-specific chaperones. Thus, Nas2 might function as a regulator of the Rpt5-CP interaction. Further analyses suggested that Nas2 has an additional function in assembly, and that mutating the tail of Rpt5 results in increased binding of the proteasome-associated protein Ecm29 to the proteasome. We showed that Ecm29 binds Rpt5 directly, thereby inducing a closed conformation of the CP substrate entry channel, and inhibiting proteasomal ATPase activity. Consistent with these activities, several proteasome mutant strains showed Ecm29-dependent accumulation of unstable substrates. Thus, Ecm29 is an inhibitor of the proteasome in vivo and in vitro. Interestingly, besides the Rpt5 mutants, several other proteasome mutants show increased levels of Ecm29, suggesting Ecm29 has a role in quality control. Consistent with this, we observed that Ecm29 associates preferably with specific mutants and nucleotide-depleted proteasomes. Based on our data we propose a model, where early in assembly Nas2 binds to the Rpt5 tail inhibiting the Rpt5-CP interaction directly. Later in assembly Ecm29 performs a quality control function, where it recognizes and remains bound to defective proteasomes. By inhibiting these proteasomes Ecm29 prevents the aberrant degradation of proteins.

Master of Science / Department of Entomology / Jeremy L. Marshall / Males providing females with gifts in order to mate is not a novel occurrence. Indeed, depending on the taxonomic system, males may provide gifts ranging from dead insects, to nutritious ejaculates to even subjecting themselves to cannibalistic feeding. Interestingly, while the burden of the costs of these gifts is primarily carried by the male, net fitness of providing or receiving such gifts must be positive for both sexes, if these gifts are to be maintained in a population. If this is not the case, and sexual conflict has exerted a cost-benefit imbalance between the sexes, then the expectation is that the system will evolve towards the reduction of nuptial gift giving. Here, utilizing the Allonemobius socius complex of crickets where females benefit greatly from cannibalizing male blood as a nuptial gift, we explore the possibility that sexual conflict is acting on gift size. To do this, we assess the gift-size distribution, and their associated fitness functions, for twelve populations that span the phylogeny and geographic distribution of this complex. We find that gift-size distributions are shifted towards small or no gifts in the majority of populations. Moreover, fitness data suggest that males providing a small or even no gift are equally successful to their large gift-giving counterparts. Taken together, the population profiles indicate that at least half of these populations are evolving towards the near or complete loss of these cannibalistic gifts – a predicted, but previously undocumented, outcome of sexual conflict. We also assess the speed at which sexual conflict is acting to alter gift size distributions in populations by comparing gift sizes between different time points in populations and across phylogenetic history. The implications of these results are discussed relative to antagonistic coevolution of behavioral traits and sexual conflict theory in general.

Doctor of Philosophy / Department of Biology / Stephen Keith Chapes / Ehrlichia chaffeensis is an obligate, intracellular bacterium that causes human monocytic ehrlichiosis (HME). The bacteria are vectored by the Lone Star tick (Amblyomma americanum), which is found primarily in the Midwestern and Southeastern United States E. chaffeensis was first reported in 1986 and HME was designated a nationally reportable disease by the United States Centers for Disease Control in 1999. Ehrlichia grows in several mammalian cell lines, but NO consensus model for pathogenesis exists for arthropods or vertebrates. Moreover, the host genes required for intracellular growth of this bacteria are unknown. We first established that the bacteria could infect and replicate both in vitro and in vivo in Drosophila melanogaster S2 cells and adult flies, respectively. We performed microarrays on S2 cells, comparing host gene expression between permissive or non-permissive conditions for E. chaffeensis growth. A total of 210 permissive, exclusive and 83 non-permissive, exclusive genes were up-regulated greater than 1.5-fold above uninfected cells. We screened flies mutant for genes identified in our microarrays for their ability to support Ehrlichia replication. Five mutant stocks were resistant to infection with Ehrlichia (genes CG6479, separation anxiety, CG3044, CG6364, and CG6543). qRT-PCR confirmed that bacterial load was decreased in mutant flies compared to wild-type controls. In particular, gene CG6364 is predicted to have uridine kinase activity. Thus, the in vivo mutation of this gene putatively disrupts the nucleotide salvage pathway, causing a decrease in bacterial replication. To further test the function of gene CG6364 in bacterial replication, we obtained cyclopentenyl cytosine (CPEC) from the National Cancer Institute. CPEC is a cytidine triphosphate (CTP) inhibitor known to deplete CTP pools in various cancers and to exhibit antiviral activity. Consequently, it inhibits de novo nucleotide synthesis, but doesn’t affect the nucleotide salvage pathway. When S2 cells were treated with CPEC and infected with Ehrlichia, an increase in bacterial replication was confirmed by qRT-PCR. Furthermore, addition of cytosine to S2 cells also resulted in increased bacterial replication. Therefore the nucleotide salvage pathway through cytidine appears necessary for bacterial replication. Our approach has successfully identified host genes that contribute to the pathogenicity of E. chaffeensis in Drosophila.

Master of Science / Department of Biology / S. K. Chapes / Ehrlichia chaffeensis is a Gram-negative, obligatorily intracytoplasmic bacterium and the causative agent of a tick-borne disease, human monocytic ehrlichiosis. In vertebrates, E. chaffeensis exhibits tropism for monocytes /macrophages. However, no clear requirements for cell tropism have been defined in ticks. Previously, our group identified two host genes that control E. chaffeensis replication in vivo in Drosophila. We used these two genes, CG6364 and separation anxiety (san) to test the hypothesis that E. chaffeensis replicates in arthropod hemocytes. Using the UAS/GAL4 RNAi system, we generated F1 flies (RNAi flies) and confirmed ubiquitous-or tissue-specific reduction in the transcript levels of the targeted genes. When RNAi flies were screened for Ehrlichia infections, we found that when either CG6364 or san were specifically suppressed in the hemocytes or in the fat body E. chaffeensis failed to replicate or cause infection. Deletion of these genes in the eyes, wings or the salivary glands did not impact fly susceptibility or bacterial replication within these organs. Our data demonstrate that in Drosophila, E. chaffeensis replicates within the hemocytes, the insect homolog of mammalian macrophages, and in the fat body, the liver homolog of mammals. This study provides insights about replication sites of E. chaffeensis in arthropods.

Master of Science / Department of Biomedical Sciences / Sanjeev K. Narayanan / Fusobacterium necrophorum is an anaerobic Gram-negative non spore forming rod shaped bacteria that is a normal inhabitant of the alimentary tract of humans and animals. Two subspecies of F. necrophorum have been recognized- subspecies necrophorum and subspecies funduliforme. Subspecies necrophorum is an opportunistic pathogen in animals causing diseases such as bovine hepatic abscesses and sheep foot rot while as subspecies funduliforme is linked with human oral and hepatic infections such as sore throats, Lemierre’s syndrome and hepatic abscesses. The pathogenic mechanisms of F. necrophorum are complex and are not well understood or defined. Several virulence factors such as leukotoxin, haemolysin, haemagglutinin and adhesin have been described. One of the most important factors in F. necrophorum bacterial pathogenesis is the adhesion of the bacteria to the host cell. The adhesion of the bacteria to the host cell helps it colonize the host tissue and this is followed by intracellular multiplication with dissemination to other tissues, which could ultimately lead to septicemia and death. Bacteria use adhesins which are proteins found in the outer membrane which help them bind with host receptors and this helps with the adhesion of the bacteria to the host cell. Not much is known about F. necrophorum adhesins. Here, we describe and characterize a novel adhesin.

Master of Science / Department of Biology / A. Lorena Passarelli / Cydia pomonella granulovirus (CpGV) is a member of the Baculoviridae family of viruses. The CpGV open reading frame 46 (CpGV-ORF46) predicts a 545 amino acid protein that shares homology with matrix metalloproteases (MMPs), a family of zinc-dependent endopeptidases that degrade extracellular matrix proteins. In silico analyses revealed the presence of putative mmp genes in all species from the Betabaculovirus genus, while no mmps were identified in members of the Alphabaculovirus, Gammabaculovirus or Deltabaculovirus genera. Unlike most cellular MMPs, baculovirus MMPs do not have a propeptide domain, a domain involved in regulating MMP activation, or a hemopexin-like domain, which is necessary for substrate binding and specificity in many MMPs. However, Betabaculovirus MMPs do contain a predicted conserved zinc-binding motif (HEXGHXXGXXHS/T) within their catalytic domain. The function of CpGV-MMP and its effects on baculovirus replication in cultured cells and insect larvae were investigated. CpGV-MMP was expressed in and purified from Escherichia coli, and activity was measured using a generic MMP substrate in vitro. CpGV-MMP had in vitro activity and its activity was specifically inhibited by MMP inhibitors. To study the effects of CpGV-MMP on virus replication and dissemination, CpGV-MMP was expressed from Autographa californica nucleopolyhedrovirus (AcMNPV) under the control of a strong and constitutive promoter, the Drosophila heat shock 70 protein promoter. Expression of CpGV-MMP did not affect virus replication in cultured cells. The effects of expressing CpGV-MMP from AcMNPV during larval infection were evaluated in the presence or absence of the AcMNPV chitinase and cathepsin genes. Insect bioassays showed that the absence of cathepsin resulted in a significant delay in larval time of death; however, this delay was compensated by expression of CpGV-MMP. In addition, larval time of death was accelerated when cathepsin, chitinase, and CpGV-MMP were all expressed. Finally, we determined the effects of CpGV-MMP on larvae melanization and liquefaction. CpGV-MMP was able to promote larvae melanization in the absence of cathepsin. CpGV-MMP, in the absence of cathepsin, was not able to promote larvae liquefaction. When chitinase was engineered to be secreted from cells, CpGV-MMP rescued liquefaction in the absence of cathepsin. In conclusion, CpGV-MMP is a functional MMP which can enhance larvae mortality with the presence of cathepsin. In addition, CpGV-MMP can promote larvae melanization; however, it can only promote liquefaction when chitinase is engineered to be secreted from cells.

Master of Science / Department of Biology / David A. Haukos / Abstract–Duckling Survival Lesser scaup (scaup) populations have been experiencing continent-wide decline since the 1980s. It is important to have complete understanding of the critical factors influencing population change (e.g., duckling survival, nesting success, and health) to advance our understanding of population dynamics and improving species conservation. Duckling survival is a primary driver of scaup demography. I conducted a capture-mark-recapture study using Cormack-Jolly-Seber models in Program MARK to compute apparent daily survival and recapture probabilities for 3256 individually marked ducklings with 620 recaptures during 2010 to 2013. The most parsimonious survival model based on a priori hypotheses found that Julian hatch date squared was the most significant predictor of survival and consistent through all four years. Mass at hatch also was significant as a quadratic effect. Duckling survival to 30 days ranged from 29.0 to 80.0. During this study, stabilizing selection played a significant role in duckling survival, which indicates that there was trade-offs for selection of an optimal timing of hatch on survival and a cost associated with hatching too early or too late and being too heavy or to light. / Abstract–Nest-Site Selection There is a hierarchical process of behavioral and environmental processes that influence habitat selection, which inherently influences the survival and fitness of that individual and contributes to population growth. I investigated nest fate, spatial attributes, and all relationships between high and low-water levels with habitat attributes (distance to upland, distance to open water, nearest neighbor distance) of located nests using general linear models in SAS, t-tests in R, and Hot Spot Analysis in ArcGIS of 481 nests over eight years. In low-water years, successful nests (X̅ = 1153 m) were located 22.0% farther from upland than unsuccessful nests (X̅ = 944 m), but support for a similar relationship was lacking in high-water years. Successful nests were located 21.0% and 23.0% (i.e., 49 and 50 m) closer to conspecific nests than unsuccessful nests in low and high-water levels, respectively. In both high and low-water level years, clusters of nests initiated later in the season coincided with Hot Spots for nest fate (i.e., high-quality habitat patches, clusters of successful nests), whereas areas that tended to be selected first, evidenced by clusters of nests initiated earlier, tended to overlap with clusters of Cold Spots for nest fate. The core Hot Spot for nest fate was in the same spot in both water level conditions and located in flooded emergent vegetation in the heart of Lower Red Rock Lake furthest from any upland habitat. Three out of six Cold Spots for nest fate both in high and low-water years were located in emergent vegetation on the perimeter of Lower Red Rock Lake adjacent to uplands. Density-dependence seems to be a factor affecting late-nesting scaup females that are apparently cuing in on the reproductive performance of conspecifics when determining where to nest. Therefore, management actions focused on survival and reproductive success of scaup should consider managing water levels and habitat for later nesting scaup to increase adult survival and ultimately recruitment of ducklings. / Abstract – Blood Parasite Prevalence Blood parasites, per se, do not lead to direct mortality, but instead reduce the health of individual birds, which may ultimately lead to decreased reproductive success. Evidence has shown that presence of blood parasites can reduce fitness, body condition, and reproductive success of waterfowl. For many avian species, the cost of reproduction is manifested as a negative relationship between female breeding effort and breeding season survival, with trade-offs occurring when these adaptive choices become detrimental to future reproductive performance. Blood was drawn for parasite load determination from 112 individual adult scaup captured from 2011to 2012 via spotlighting and drive-trapping. Parasite prevalence was determined through blood assays that were created using a two-slide wedge technique. Relationships among seasonal heterophile:lymphocyte ratio (a proxy for health), body mass at time of capture (throughout pre-, during, and post-breeding periods), breeding status (females only), and Julian date of capture (date of capture) of capture with parasite prevalence were analyzed using linear (lm) regression models in R 2.15.2. The blood parasite infection rate was 5.0%, with prevalence differing by gender with 33.3% of males positive for blood parasites compared to 1.0% of females. The presence of blood parasites did not affect health, fitness, or breeding status of scaup. A quadratic relationship was found with body mass and date of capture, indicating that body mass increased from pre-breeding period to the breeding period and decreased significantly at the end of the summer during molt. A negative relationship between the heterophile:lymphocyte ratio of female scaup and date of capture (i.e., the health of scaup females was greatest during the pre-breeding period after which it consistently decreased until the molting period). A strong negative correlation between heterophile:lymphocyte ratio and body mass was found in both genders, which indicated that scaup in poor body condition were also in poor health at the end of the breeding season.

Doctor of Philosophy / Division of Biology / Theodore Morgan / Temperature is a critical environmental parameter and thermal variation has significant effects on local adaptation and species distributions in nature. This is especially true for organisms that are isothermal with their environment. Variation in temperature imposes stress and directly influences physiology, behavior, and fitness. Thus, to thrive across a range of thermal environments populations must contain sufficient genetic variation, the capacity to respond plastically, or some combination of both genetic and plastic responses. In this work I first quantified patterns of phenotypic and genetic variation in nature and then dissected the genetic basis of variation in thermal traits. In the first aim I used natural populations of Drosophila melanogaster collected from a latitudinal transect in Argentina to investigate variation in heat stress resistance and cold plasticity within and among populations. I found heat stress resistance was highly variable within populations, but was strongly associated with the monthly maximum average temperature of each site. For cold plasticity I was able to demonstrate significant variation in plasticity within and among populations, however the among population variation was best explained by the altitude of each site. I hypothesized that this was caused by a difference in temperature fluctuations at high altitude sites relative to low altitude sites. To evaluate this hypothesis I paired our study with existing laboratory data that demonstrated significant fitness differences between high and low plasticity (and altitude) sites when these populations were reared in variable thermal environments. Thus, cold plasticity is an adaptive response to environmental variation. The final project focused on understanding the genetic basis of thermal variation. I fine-mapped a single co-localized heat and cold tolerance QTL via deficiency and mutant complementation mapping to identify four novel thermal candidate genes. There was no overlap of the deficiencies or genes associated with cold or heat stress resistance. Sequence analysis of each gene identified the polymorphisms that differentiate the lines. To test for independent associations between these polymorphisms and variation in nature the Drosophila Genome Reference Panel was used to confirm associations between allelic variation and cold tolerance in nature.