Department of Biology

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These digital collections include theses, dissertations, faculty publications, and datasets from the Department of Biology.

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Open Access
A new woody perspective on copper homeostasis: systemic copper transport and distribution, effect of copper on lignification, and water transport in hybrid poplar
(Colorado State University. Libraries, 2022) Hunter, Cameron Ross, author; Pilon, Marinus, advisor; Gleason, Sean, advisor; Pilon-Smits, Elizabeth, committee member; Argueso, Cristiana, committee member; Bush, Daniel, committee member
Copper (Cu) is an essential micronutrient for plants. Chapter 1, as background for this dissertation, reviews the functions and homeostasis of Cu. We know at the cellular level how Cu is delivered to target proteins in the chloroplasts, thus explaining in a large part why Cu deficient plants have reduced photosynthetic capacity. However, Cu is also a cofactor of lignin polymerization enzymes that affect cell wall and xylem structures required for water and mineral transport. How Cu deficiency affects water transport, mineral nutrition, and photosynthesis at a whole plant level is underexplored. To address this knowledge gap, we used hybrid white poplar as a model. In chapter 2, a stable isotope method to trace Cu movement in poplar tissues was coupled with analysis of photosynthesis and stomatal conductance. Upon resupply of Cu, priority targets identified were stems and younger leaves which recovered quickly and was associated with higher stomatal conductance. In chapter 3, the effect of Cu deficiency on the elemental composition of leaves and stems of different age were analyzed. Interestingly, tissue type and age, as well as Cu deficiency, were found to all significantly affect within-plant nutrient partitioning patterns. In chapter 4, the effects of Cu deficiency on cell wall chemical composition and water transport traits were determined. Although Cu deficiency strongly affected cell wall chemistry, it did not significantly impact hydraulic capacity nor the density and size of xylem vessels in stems. However, Cu deficiency resulted in markedly stiffer mesophyll cell walls, possibly arising from changes to cell wall chemistry or structure. Together, these results, as discussed in chapter 5, indicate that although xylem lignification was adversely affected by Cu deficiency, the water transporting vessels remained largely unaffected, thus allowing efficient recovery. This work opens new avenues to explore the effects of plant nutrition on whole-plant physiology and function.
Open Access
A prickly puzzle: phylogeny and evolution of the Carduus-Cirsium group (Cardueae: Compositae), and untangling the taxonomy of Cirsium in North America
(Colorado State University. Libraries, 2020) Ackerfield, Jennifer R., author; Simmons, Mark, advisor; Kondratieff, Boris, committee member; Smith, Melinda, committee member; Steingraeber, David, committee member; Funk, Vicki, committee member
Generic delimitations within the cosmopolitan Carduus-Cirsium group (i.e., "thistles") have a long history of taxonomic confusion and debate. We present the most comprehensive molecular phylogeny of the group to date to test generic limits, reconstruct the evolution of pappus type, and elucidate the role of chromosomal evolution. We offer two solutions for the recognition of monophyletic genera: (1) consolidate all taxa into one large genus (Carduus or Cirsium), or (2) recognize each major clade as a genus (Carduus, Cirsium, Eriolepis, Notobasis, Picnomon, Silybum, and Tyrimnus). Under the second proposal, the cryptic genus Eriolepis is segregated from Cirsium, and the African Carduus are included within Cirsium. The best diagnosable morphological character to delimit the genera is pollen type, which is not practical in field-based application. We caution that prior to implementing either solution, a thorough, comprehensive morphological analysis of all current members of Cirsium sect. Epitrachys (= genus Eriolepis) be completed. Future morphological studies may find additional achene or leaf surface characters that could be used for practical field identification of the segregate genera. The data show that the plumose pappus state is symplesiomorphic for the group, with one transition to barbellate pappus, likely followed by a reversal to its ancestral state as the group colonized Eurasia. The data are consistent with a North African origin in the region of the Mediterranean and a single colonization event to North America. An ancestral chromosome state of n = 17 is hypothesized for the group, and a descending dysploidy series in Carduus is hypothesized to correspond with the aridification of the Mediterranean region. The Carduus-Cirsium group highlights the difficulty of delimiting morphologically similar, cryptic genera. Cirsium is one of the most taxonomically challenging groups of Compositae in North America. This study represents the first attempt to infer a broadly sampled phylogeny of Cirsium in North America. The two main objectives are to: (1) test whether currently hypothesized species variety complexes (C. arizonicum, C. clavatum, C. eatonii, and C. scariosum) constitute monophyletic lineages, and (2) recircumscribe any taxa that are identified as problematic. Phylogeny reconstructions based on DNA sequence data from two nuclear ribosomal regions and four plastid markers were used to infer evolutionary lineages and test species' delimitations. Eight species varietal complexes were resolved as polyphyletic. We recircumscribed these complexes and in doing so found evidence to support the recognition of six new taxa. We hypothesize that the extensive taxonomic difficulty within Cirsium is the result of several factors: 1) previously undescribed taxa, 2) inadequate representation of taxa from herbarium specimens, 3) phenotypic convergence, 4) hybridization, and 5) incipient speciation. While we can provide evidence to support the recircumscription of some taxa, others remain unresolved.
Open Access
A study of plant domestication and evolution through the taxonomic revision of wild North American Humulus, a phytochemical assay for stimulant alkaloids in Celastraceae, and a phylogeographic analysis of Catha edulis in areas of historic cultivation
(Colorado State University. Libraries, 2015) Tembrock, Luke Raymond, author; Simmons, Mark P., advisor; Angert, Amy L., committee member; Richards, Christopher M., committee member; Ward, Sarah M., committee member
The cultivation of plant species is essential to the survival of humans. The process of artificial selection that is used to modify wild individuals into improved cultivars results in genetic and morphological changes from wild progenitors. In order to understand the evolutionary patterns and processes involved with artificial selection both wild and cultivated populations must be thoroughly studied. Numerous methods are used to study the process of evolution under cultivation such as biology, chemistry, geography, history, linguistics and archeology. The understanding of evolution in a crop species is essential in current improvement programs to increase yield for a given crop. I employed methods from the fields of taxonomy, analytical chemistry and phylogeography to study the process of evolution in cultivated plant species and/or their wild relatives. From a review of taxonomic, genetic, and phytochemical literature, as well as examination of morphological features I revised the wild North American Humulus (Cannabacae) in a manner that properly delimits the diversity found among the North American species. Using GC–MS and a forensics based derivatization method I assayed for the stimulant alkaloids cathinone, cathine, and similar compounds across the Celastraceae plant family. It was found that that qat (Catha edulis) was the only species of those tested that biosynthesized cathinone and cathine. Using phylogeographic and population genetic techniques I inferred three wild regional origins, hybridization and numerous translocations out of the centers of origin for cultivated qat. From farmer interviews I examined what properties, genotype, phenotype, and/or geography explained the naming convention for qat cultivars among qat farmers. The character of stem color was found to highly plastic and thus genotype was not significantly correlated with the naming convention. Geographic patterns were confirmed for several cultivar names suggesting that anthropogenic factors are important in the naming conventions used among qat farmers. These four separate studies provide findings that not only clarify our understanding of evolutionary patterns among wild and cultivated species but provide a framework for breeding, conservation and forensic applications in the future.
Open Access
A survey of foliar fungal endophyte communities of Rocky Mountain bristlecone pine populations in the Colorado Rocky Mountains
(Colorado State University. Libraries, 2017) Albertson, Alyssa, author; Bedinger, Patricia, advisor; Kassenbrock, C. Kenneth, advisor; Stewart, Jane E., committee member; Schoettle, Anna W., committee member
Rocky Mountain bristlecone pine (Pinus aristata) is an exceptionally long-lived charismatic tree species found at high elevations in the southern Rocky Mountains of Colorado, New Mexico, and Arizona (Fryer, 2004). This species has recently come under threat from the disease white pine blister rust (WPBR). White pine blister rust is caused by the pathogenic fungus Cronartium ribicola, which was inadvertently introduced into North America from Europe in the early 1900's, and has since spread widely with devastating impacts (Burns et al., 2008). In North America, WPBR is largely lethal to five-needle pine species. In Colorado, WPBR has been found in stands of Rocky Mountain bristlecone pine and limber pine (Pinus flexilis), and efforts have been made to identify trees with increased resistance to the disease. The USDA Forest Service Rocky Mountain Research Station has identified specific trees that harbor some level of heritable resistance to WPBR, versus those appearing fully susceptible (Schoettle, 2004; Schoettle et al., 2012; Schoettle et al., 2014). Essentially all plants in the wild harbor endophytic bacteria and fungi, which are defined as co-existing in plant tissues without causing evidence of disease, and it is increasingly appreciated that endophytes can alter plant responses to both biotic and abiotic stresses (Rodriguez et al., 2008). It has been reported that fungal endophytes can enhance resistance to blister rust in western white pine (Pinus monticola) (Ganley et al., 2008). The endophytic fungi of Rocky Mountain bristlecone pines have not been previously studied. We used two techniques to survey the endophytic fungal communities present in wild populations of resistant and susceptible bristlecone pines. The first technique was to isolate endophytic fungi by culturing surface-sterilized pine needles. The second technique was to extract DNA from the pine needles, and use PCR amplification of fungal-specific sequences, followed by high-throughput Next Generation sequencing (NGS) to identify and quantify fungi present, regardless of whether or not they could be cultured. By culture we recovered 259 fungal isolates that were placed into 81 morphological groups, whereas the NGS returned 42,003 useable DNA sequences that were grouped into 791 operational taxonomic units (OTUs). The two techniques used in this study had significant overlap; most of the cultured fungi were also identified in the NGS data set. The high throughput sequencing data also revealed differences between the endophyte populations of trees previously inferred to have or lack resistance to white pine blister rust at each location. Further research will be needed to understand whether endophytes may modify, slow, or even prevent infection by the pathogen C. ribicola.
Open Access
Advancing conservation genomics of migratory species toward a full annual cycle approach
(Colorado State University. Libraries, 2023) DeSaix, Matthew G., author; Ruegg, Kristen C., advisor; Funk, W. Chris, committee member; Koons, David N., committee member; Marra, Peter M., committee member
Global biodiversity loss is one of the foremost concerns of conservation efforts in the 21st century. The maintenance of genetic diversity within species is a critical factor in a species' persistence and adaptive potential in the face of changing environmental conditions. Migratory species make up more than 12% of the global vertebrate biodiversity and pose distinct challenges to conservation efforts due to inhabiting different geographical regions at different times of the year. The field of conservation genomics provides a valuable toolkit to addressing and understanding global biodiversity loss but requires additional methodological developments to better address the conservation challenges posed by migratory species. In my dissertation, I demonstrate advancements in conservation genomics aimed toward better understanding migratory species. In my first study, I addressed the question of ecological and genomic vulnerability to climate change in the Brown-capped Rosy-Finch (Leucosticte australis), an elevational migratory songbird of conservation concern. Second, I addressed a methodological gap in population genomics and developed statistical genetics models for using genotype likelihood data from low-coverage whole genome sequencing data to implement population assignment. In my last study, I demonstrate the utility of low-coverage whole genome sequencing for population assignment with detailing migratory connectivity in the American Redstart (Setophaga ruticilla). Altogether, my doctoral research demonstrates how genomic tools can help unravel the complexities of migratory species conservation. Furthermore, the species-specific results are tied to knowledge gaps identified by wildlife managers and provide valuable information tied to conservation and management applications.
Open Access
Altered behavior and cost of manipulation: the acanthocephalan Leptorhyncoides thecatus in its amphipod host Hyalella azteca
(Colorado State University. Libraries, 2014) Stone, Charles F., author; Moore, Janice, advisor; Kondratieff, Boris, committee member; Ghalambor, Cameron, committee member; Naug, Dhruba, committee member
Behavioral manipulation occurs when a parasite causes changes in its host's behavior to the parasite's benefit. The parasite benefits from these behavioral changes by increased survival or transmission. It has been hypothesized that such manipulation carries a cost for the parasite because energy allocated to manipulation does not contribute to growth or reproduction. The acanthocephalan parasite Leptorhynchoides thecatus provides a system in which to test this concept. This parasite uses the amphipod Hyalella azteca as an intermediate host and fish as definitive hosts; it has not been previously shown to alter host behavior. This system is advantageous for testing costs of manipulation: the size of the larval cystacanth stage in the intermediate host provides an easily quantified measure of fitness. Larger cystacanths establish in the fish host more frequently than smaller cystacanths. If manipulation is costly, I predict that there should be a negative relationship between the strength of behavioral change and fitness measures (larval size). I compared geotaxis, phototaxis, photophilia, and activity responses of infected and uninfected H. azteca to determine whether L. thecatus modified behavior. I also measured the responses of infected and uninfected amphipods to alarm pheromones and predator kairomones. I then investigated whether these behavioral changes were correlated with larval size. I found that L. thecatus does indeed alter host behavior. Compared to uninfected amphipods, infected amphipods were found higher in the water column, spent greater time in lighted areas, and were more active. There was no difference in phototaxis; both groups of amphipods swam away from a direct light source. Infected amphipods also reduced anti-predator responses to alarm pheromones and predator kairomones. This is the first example of altered alarm pheromones behavior in parasitized amphipods. These findings strongly suggest that L. thecatus increases encounters between its intermediate host and definitive host predators and that the parasite increases its transmission rate through behavioral manipulation. None of these behavioral changes were correlated with a decrease in larval size as predicted by the manipulation cost hypothesis. In fact, larger cystacanths altered geotaxis and photophilia more than smaller cystacanths did. Finally, I compare L. thecatus host use data collected from Atkinson Reservoir, Nebraska, between 2008 and 2011 to published data from 1979-1980. Both data sets show that the Green Sunfish (Lepomis cyanellus) and Pumpkinseed Sunfish (Lepomis gibbosus) are the highest quality hosts for this population. However, the current data suggest a possible shift in secondary hosts from Largemouth Bass (Micropterus salmoides) to Bluegill Sunfish (Lepomis macrochirus). Understanding the cost associated with any trait sheds light on the evolution and maintenance of that trait. This dissertation uses a unique population of L. thecatus to add this parasite to the growing list of those that behaviorally manipulate their hosts, and to demonstrate that, contrary to predictions from the theoretical literature, behavioral manipulation is not necessarily costly.
Open Access
Alternative splicing and its regulatory mechanisms in photosynthetic eukaryotes
(Colorado State University. Libraries, 2011) Link, Alicia, author; Reddy, A. S. N., advisor; Stack, Stephen, committee member; Lapitan, Nora, committee member
In recent years, alternative splicing (AS) of pre-mRNAs, which generates multiple transcripts from a single gene, has emerged as an important process in general proteome diversity and in regulatory gene expression in multicellular eukaryotes. In Arabidopsis over 40% of intron-containing genes are alternatively spliced. However, mechanisms by which AS is regulated in plants are not fully understood, primarily due to the lack of an in vitro splicing system derived from plants. Furthermore, the extent of AS in simple unicellular photosynthetic eukaryotes from which plants have evolved is also not known. My research addresses these two attributes of splicing in plants. In Part 1 of my thesis, I have investigated an aspect of AS regulation in plants. We have previously shown that an SR-related splicing regulator called SR45 regulates AS of pre-mRNAs in Arabidopsis by altering splice site selection (Ali et al. 2007). In this work using bimolecular fluorescent complements, I have demonstrated that SR45 interacts with U2AF35, an important spliceosomal protein involved in 3' splice site selection in plant cells. This interaction takes place in the nucleus, specifically in the subnuclear domains called speckles, which are known to contain splicing regulators and other proteins involved in transcription. My work has shown that SR45 interacts with both paralogs of U2AF35 and I mapped the domains in SR45 that are involved in its interaction with U2AF35. In addition, my studies have revealed interaction of the paralogs as hetero- and homodimers. Interestingly, U2AF35 was found to interact with U1-70K, a key protein involved in 5' splice site selection. Based on this work and previous work in our laboratory, a model is proposed that explains the role of SR45 in splice site selection. In the second part of my work I studied the extent of alternative splicing (AS) in the unicellular green alga Chlamydomonas, that shares a common ancestor with land plants. In collaboration with Dr. Asa Ben Hur's lab, we have performed a comprehensive analysis of AS in Chlamydomonas reinhardtii using both computational and experimental methods. Our results show that AS is common in Chlamydomonas, but its extent is less than what is observed in land plants. However, the relative frequency of different splicing events in Chlamydomonas is very similar to higher plants. We have found that a large number of genes undergo alternative splicing, and together with the simplicity of the system and the use of available molecular and genetic tools. This organism is an experimental system to investigate the mechanisms involved in alternative splicing. To further validate predicted splice variants, we performed extensive analysis of AS for two genes, which not only confirmed predictions but also revealed novel splice variants, suggesting that the extent of AS is higher than we predicted. AS can also play a role in the regulation of gene expression through processes such as regulated unproductive splicing and translation (RUST) that involves nonsense-mediated decay (NMD), a mechanism of mRNA surveillance that degrades transcripts containing premature termination codons (PTCs). The basic mechanism of NMD relies upon many factors, but there are three critical proteins, termed the UP-frameshift (UPF) proteins due to their ability to up-regulate suppression of nonsense transcripts. UPF1, UPF2, and UPF3 appear to be conserved across animals and plants. Our analysis of AS has found that in Chlamydomonas, many splice variants have a premature termination codon (PTC). However, to date, the mechanism of NMD has not been investigated in Chlamydomonas. Analysis of the Chlamydomonas genome sequence shows that UPF1, 2, and 3 proteins are present, and we have shown that they share some sequence similarity with both plants and humans, indicating that the process of NMD may be present in this organism. To address the role of UPFs in NMD in Chlamydomonas, we have utilized the artificial miRNA approach. I have generated stably transformed Chlamydomonas cell lines that are expressing amiRNA for UPF1 and UPF3 that will be useful in analyzing NMD of selected genes as well as all PTC-containing transcripts globally.
Open Access
Analyzing genetic response mechanisms associated with copper homeostasis in Populus trichocarpa using a bioinformatics approach
(Colorado State University. Libraries, 2013) Patterson, Eric, author; Pilon, Marinus, advisor; Bedinger, Patricia, committee member; Jahn, Courtney, committee member; Walters, Christina, committee member
Copper is an essential micronutrient for plants and plays an important role in photosynthesis, respiration, hormone signaling, cell wall structure and wound healing. Copper deficiency can cause chlorosis, leaf curling, and weakened stems. It is proposed that under copper deficient conditions plants down regulate genes whose proteins use copper as a cofactor but also play an "unessential" role for the plants survival, thereby preserving copper for more "essential" proteins like plastocyanin or cytochrome-C oxidase. Down-regulation of "unessential" genes is performed by the copper microRNAs miR307, miR398, and miR408. This thesis increases our understanding of copper homeostasis in plants by analyzing the transcriptomic response of Populus trichocarpa to copper deficiency in four vegetative organs and applies this knowledge to the study of multi-copper oxidases. Organs have drastically different responses to copper deficiency with few genes being systemically differentially expressed and most genes that are differential expressed only are in one organ. Our data also show that not all genes are regulated to the same extent. Genes that are already highly expressed (>50 RPKM) under copper-sufficient conditions are only up-regulated 1- to 4-fold, while low expressed genes can be up-regulated as much as 8-fold. We go on to describe 25 unannotated genes as laccases based on their sequence similarity with known laccases from Arabidopsis and Populus. The laccases break up into seven phylogenetically distinct groups. Each of the seven groups have a distinct expression pattern across the four organs in response to copper deficiency that seems to be mediated by Cu-miRNAs miR397 and miR408.
Open Access
Aquatic insect responses to predation and temperature: variation in context dependent trophic interactions
(Colorado State University. Libraries, 2017) Morton, Scott Gregory, author; Poff, N. LeRoy, advisor; Kondratieff, Boris, committee member; Schmidt, Travis, committee member
Trophic cascades, the indirect effects of carnivores on primary producers mediated by herbivores, remains a central theme of ecological theory. How climate change will alter the mechanisms controlling such interactions remains largely unexplored, certainly in stream ecosystems. In montane streams, stonefly predators have been documented to indirectly affect algal biomass by influencing the distribution, abundance, behavior, and life histories of invertebrate grazers. Density mediated indirect interactions (DMII) occur when primary producer biomass is primarily influenced by changes in herbivore abundance due to consumption by predators. Trait-mediated indirect interactions (TMII) alter primary producer abundance through non-consumptive interactions such as anti-predatory behaviors. In this research, I conducted mesocosm experiments on stonefly predators and mayfly prey to determine the relative importance of grazers on regulating algal production under three temperature treatments intended to simulate climate warming. Furthermore, I examined the influence of both DMII and TMII on algal production through consumptive and non-consumptive predatory treatments. I found algal biomass to decrease as temperature increased, however found no differences among grazer-alone treatments versus DMII or TMII on algal production.
Open Access
Arabidopsis thaliana VOZ (Vascular plant One-Zinc finger) transcription factors are required for proper regulation of flowering time
(Colorado State University. Libraries, 2013-04-15) Celesnik, Helena, author; Ali, Gul S., author; Robison, Faith M., author; Reddy, Anireddy S. N., author; The Company of Biologists Ltd., publisher
Transition to flowering in plants is tightly controlled by environmental cues, which regulate the photoperiod and vernalization pathways, and endogenous signals, which mediate the autonomous and gibberellin pathways. In this work, we investigated the role of two Zn2+-finger transcription factors, the paralogues AtVOZ1 and AtVOZ2, in Arabidopsis thaliana flowering. Single atvoz1-1 and atvoz2-1 mutants showed no significant phenotypes as compared to wild type. However, atvoz1-1 atvoz2-1 double mutant plants exhibited several phenotypes characteristic of flowering-time mutants. The double mutant displayed a severe delay in flowering, together with additional pleiotropic phenotypes. Late flowering correlated with elevated expression of FLOWERING LOCUS C (FLC), which encodes a potent floral repressor, and decreased expression of its target, the floral promoter FD. Vernalization rescued delayed flowering of atvoz1-1 atvoz2-1 and reversed elevated FLC levels. Accumulation of FLC transcripts in atvoz1-1 atvoz2-1 correlated with increased expression of several FLC activators, including components of the PAF1 and SWR1 chromatin-modifying complexes. Additionally, AtVOZs were shown to bind the promoter of MOS3/SAR3 and directly regulate expression of this nuclear pore protein, which is known to participate in the regulation of flowering time, suggesting that AtVOZs exert at least some of their flowering regulation by influencing the nuclear pore function. Complementation of atvoz1-1 atvoz2-1 with AtVOZ2 reversed all double mutant phenotypes, confirming that the observed morphological and molecular changes arise from the absence of functional AtVOZ proteins, and validating the functional redundancy between AtVOZ1 and AtVOZ2.
Open Access
Are you what you eat? Assessing intracellular effects of supplemental lipid within mouse (C2C12) skeletal muscle cells
(Colorado State University. Libraries, 2021) Montano, Dominique, author; Kanatous, Shane, advisor; Bell, Christopher, advisor; Engle, Terry, committee member; Florant, Gregory, committee member
The expression of myoglobin, an oxygen-binding protein, facilitates aerobically-powered dives by effectively distributing a finite oxygen supply during breath-hold dives in marine mammals (Costa 2004, Kanatous et al. 1999, Wright 2006). Multiple factors have been experimentally shown to contribute to increasing myoglobin expression, thus influencing aerobic capacity. Among the known expression pathways, the most recent and least studied involves a link between lipid supplementation and increased myoglobin expression. We are unaware of the specific conditions that drive this expression, and whether or not fatty acids play an intracellular role in upregulating myoglobin. This work elucidates the effects of lipid supplementation on the expression of myoglobin in terrestrial mammals, as well as enhances our understanding of cues that initiate the expression of factors contributing to the positive development of aerobically-based exercise in the whole animal. In order to understand these lipid influences on an aerobically-adapted animal such as a marine mammal, we focused on developing experimental designs and applications geared towards a model mammalian system of C2C12 mouse cells. Lipids possess the capacity to influence aerobic ability in mammalian models, whether through marine mammals' reliance on lipid metabolism to power aerobically-based exercise, regulating mitochondrial respiration, fatty acid metabolism that generates increased ATP from oxidative phosphorylation relative to metabolic pathways like glycolysis, lipids acting as ligands to affect genes that contribute to aerobic capacity, or lipid supplementation that increases myoglobin expression. Multiple studies from our lab have shown that lipid present in the media upregulates myoglobin in C2C12 cells, driving expression of the protein in conjunction with other elements. Although we have determined a link between lipid supplementation and myoglobin expression, the particular intracellular effects ranging from uptake, transporter protein regulation, and either storage, metabolism, or specific effects directly affecting myoglobin expression still remain to be uncovered. The factors and conditions that regulate myoglobin expression are extensive, and lipids as an avenue to myoglobin expression is a relatively new exploration that will have applications for numerous areas of physiological research. This work has applications in multiple mammalian models, including humans, and will serve to enhance our understanding of the factors that drive aerobically-based endurance exercise, as well as understanding the cues and stimuli required to increase expression of key factors that regulate aerobic capacity in mammalian systems.
Open Access
Behavioral alteration in the honeybee due to parasite-induced energetic stress
(Colorado State University. Libraries, 2012) Mayack, Christopher, author; Naug, Dhruba, advisor; Kondratieff, Boris, committee member; Stephens, David, committee member; Moore, Janice, committee member
Parasites are dependent on their hosts for energy and honeybee foragers with their high metabolic demand due to flight are especially prone to an energetic stress when they are infected. The microsporidian gut parasite Nosema ceranae is relatively new to the honeybee, Apis mellifera and because it is less co-evolved with its new host the virulence from infection can be particularly high. Using a series of feeding and survival experiments, I found that bees infected with N. ceranae have a higher appetite and hunger level, and the survival of infected bees is compromised when they are fed with a limited amount of food. However, if fed ad libitum the survival of infected individuals is not different from that of uninfected bees, demonstrating that energetic stress is the primary cause of the shortened lifespan observed in infected bees. I then developed a high throughput colorimetric assay to analyze hemolymph sugar levels of individual bees to demonstrate that the parasite mediated energetic stress is expressed as lower trehalose levels in free-flying bees, which suggests that infected bees are not only likely to have a reduced flight capacity but they are also unable to compensate for their lower energetic state. One of the ways in which the changing energetic state of an individual is predicted to impact its behavior is its sensitivity to risk although this has never been convincingly demonstrated. According to the energy budget rule of Risk Sensitivity Theory, it is adaptive for an animal to be risk averse when it is on a positive energy budget and be risk prone when it is on a negative budget because the utility of a potential large reward is much higher in the latter case. By constructing an empirical utility curve and conducting choice tests using a Proboscis Extension Response assay in bees that have been variously manipulated with respect to their energy budgets, I comprehensively demonstrated that bees shift between risk averse to risk prone behavior in accordance with the energy budge rule. Even more importantly, I showed that this shift is contingent upon a change in the energy budget as bees maintained on constant high or low energy budgets were found to be risk indifferent. Given that Nosema infected bees have been seen to forage precociously and inclement weather, my results suggest that such risky foraging might be a consequence of the lower energetic state of infected foragers. As these previous results suggest that parasitism, by lowering their energetic state could have a significant influence on how infected bees forage, I decided to test if the energetic state of an individual can regulate its foraging independent of the colony level regulation of foraging. I uncoupled the energetic state of the individual from that of the colony by feeding individual bees with the non-metabolizable sugar sorbose, thereby creating hungry bees in a satiated colony. I found that these energy depleted bees initially compensate for their lower energetic state by being less active within the colony and taking fewer foraging trips, but not by feeding more within the colony. However, with further depletion in their energetic state, these bees increase their foraging frequency showing that foraging is still partly regulated at the individual level even in a eusocial animal such as the honeybee. My research therefore shows that the energetic stress from a parasite could be a general mechanism that leads to significant behavioral alterations in infected individuals. Since the energetic state of an animal is a fundamental driver of its behavior, such a mechanism underlying behavioral alterations could have a significant impact on the life history of the host and transmission dynamics of a disease. More specifically, these results also suggest that a parasitic infection leading to energy depleted bees going out to forage in a risky manner also provides a plausible mechanism that explains the recent observations of bees disappearing from their colonies.
Open Access
Behavioral responses associated with a human-mediated predator shelter
(Colorado State University. Libraries, 2014-04) Graeme, Shannon, author; Cordes, Line S., author; Hardy, Amanda R., author; Angeloni, Lisa M., author; Crooks, Kevin R., author; Public Library of Science, publisher
Human activities in protected areas can affect wildlife populations in a similar manner to predation risk, causing increases in movement and vigilance, shifts in habitat use and changes in group size. Nevertheless, recent evidence indicates that in certain situations ungulate species may actually utilize areas associated with higher levels of human presence as a potential refuge from disturbance-sensitive predators. We now use four-years of behavioral activity budget data collected from pronghorn (Antilocapra americana) and elk (Cervus elephus) in Grand Teton National Park, USA to test whether predictable patterns of human presence can provide a shelter from predatory risk. Daily behavioral scans were conducted along two parallel sections of road that differed in traffic volume - with the main Teton Park Road experiencing vehicle use that was approximately thirty-fold greater than the River Road. At the busier Teton Park Road, both species of ungulate engaged in higher levels of feeding (27% increase in the proportion of pronghorn feeding and 21% increase for elk), lower levels of alert behavior (18% decrease for pronghorn and 9% decrease for elk) and formed smaller groups. These responses are commonly associated with reduced predatory threat. Pronghorn also exhibited a 30% increase in the proportion of individuals moving at the River Road as would be expected under greater exposure to predation risk. Our findings concur with the 'predator shelter hypothesis', suggesting that ungulates in GTNP use human presence as a potential refuge from predation risk, adjusting their behavior accordingly. Human activity has the potential to alter predator-prey interactions and drive trophic-mediated effects that could ultimately impact ecosystem function and biodiversity.
Open Access
Body size, first year breeding, and extra-pair paternity in an island endemic, the Island Scrub-Jay
(Colorado State University. Libraries, 2014) Desrosiers, Michelle A., author; Angeloni, Lisa M., advisor; Ghalambor, Cameron K., advisor; Funk, W. Chris, committee member; Sillett, T. Scott, committee member; Crooks, Kevin R., committee member
Body size is a fundamental characteristic that shapes all aspects of an organism's biology. The advantages of large body size may include increased probability of territory acquisition, a critical component of fitness for species that require a territory to breed. Large body size, an indicator of quality, may also be advantageous to males in acquiring mates, including matings outside of a pair bond. Such advantages could be especially important in island systems because habitat saturation may result in strong intra-specific competition for territories, and females may be especially motivated to seek large extra-pair mates to increase the body size of their offspring. We tested the role of body size in determining the ability of an island-endemic bird, the Island Scrub-Jay, Aphelocoma insularis, to acquire a territory and breed in their first spring, as well as to sire extra-pair offspring. We compared the body size of individuals that obtained a territory and bred to those that did not, as well as the body size of social fathers to the extra-pair sires to whom they lost paternity. We found that large body size was important in the siring of extra-pair young. However, body size did not predict the ability of male or female Island Scrub-Jays to acquire a territory and breed in their first year. We suggest that year-to-year variation in environmental conditions and chance may be more important than a large body size or weapon performance in early territory acquisition. Our study provides evidence for a mechanism, specifically female preference for a large body size in males, that supports the observed rates of extra-pair paternity, and demonstrates the general difficulty, even for individuals with a relatively large body size, of acquiring a territory as a yearling in an island system with saturated habitat.
Open Access
Candidate reservoir underlying re-emergent plague outbreaks
(Colorado State University. Libraries, 2019) Markman, David W., author; Antolin, Michael F., advisor; von Fischer, Joseph C., committee member; Gage, Kenneth L., committee member; Bowen, Richard A., committee member
To view the abstract, please see the full text of the document.
Open Access
Carbon-degrading enzyme activities stimulated by increased nutrient availability in Arctic tundra soils
(Colorado State University. Libraries, 2013-10) Koyama, Akihiro, author; Wallenstein, Matthw D., author; Simpson, Rodney T., author; Moore, John C., author; Public Library of Science, publisher
Climate-induced warming of the Arctic tundra is expected to increase nutrient availability to soil microbes, which in turn may accelerate soil organic matter (SOM) decomposition. We increased nutrient availability via fertilization to investigate the microbial response via soil enzyme activities. Specifically, we measured potential activities of seven enzymes at four temperatures in three soil profiles (organic, organic/mineral interface, and mineral) from untreated native soils and from soils which had been fertilized with nitrogen (N) and phosphorus (P) since 1989 (23 years) and 2006 (six years). Fertilized plots within the 1989 site received annual additions of 10 g N⋅m-2⋅year-1 and 5 g P⋅m-2⋅year-1. Within the 2006 site, two fertilizer regimes were established - one in which plots received 5 g N⋅m-2⋅year-1 and 2.5 g P⋅m-2⋅year-1 and one in which plots received 10 g N⋅m-2⋅year-1 and 5 g P⋅m-2⋅year-1. The fertilization treatments increased activities of enzymes hydrolyzing carbon (C)-rich compounds but decreased phosphatase activities, especially in the organic soils. Activities of two enzymes that degrade N-rich compounds were not affected by the fertilization treatments. The fertilization treatments increased ratios of enzyme activities degrading C-rich compounds to those for N-rich compounds or phosphate, which could lead to changes in SOM chemistry over the long term and to losses of soil C. Accelerated SOM decomposition caused by increased nutrient availability could significantly offset predicted increased C fixation via stimulated net primary productivity in Arctic tundra ecosystems.
Open Access
Cattle use of prairie dog towns on the Shortgrass Steppe of Colorado
(Colorado State University. Libraries, 2000) Guenther, Debra A., author; Detling, James K., advisor; Rittenhouse, L. R. (Larry Ronald), 1940-, committee member; Steingraeber, David Allen, 1953-, committee member
Studies on the mixed-grass prairie have found that native large herbivores preferentially graze on prairie dog towns. I investigated the use of prairie dog towns by cattle (Bos taurus) in northeast Colorado by conducting surveys of cattle and vegetation on the Shortgrass Steppe Long Term Ecological Research Site from June-August, 1999. Twelve pastures containing 15 black-tailed prairie dog (Cynomys ludovicianus) towns were surveyed three times a week, and the number of cattle on the towns and their behavior were recorded. A subset of three pastures was intensively surveyed twice weekly wherein the habitat and activity of a randomly chosen focal animal was recorded every six minutes for 3.5 hours. Bite and step counts of other individuals were recorded for five-minute intervals. Vegetation height and cover data were collected monthly on each of the six habitat types. Resource selection functions for driving survey data indicated no significant difference between prairie dog town use and availability. Regression analysis showed no correlation between rain events and prairie dog town use by cattle. The intensively surveyed pastures yielded similar results: i.e., cattle did not significantly prefer or avoid the prairie dog towns. Cattle spent 60% of their time grazing, 13% resting, and 15% traveling when present on prairie dog towns. These percentages were not significantly different from most other habitat types. There were no significant differences in the number of bites per step taken by cattle on towns compared to off town swales, Atriplex canescens terraces, and crested wheatgrass (Agropyron cristatum) strips. Five plant species palatable to cattle occurred in relatively high frequencies (36-65%) on prairie dog towns. In general, bare ground, litter, and vegetation cover on prairie dog towns did not significantly differ from most other habitat types. Vegetation on prairie dog towns was however significantly shorter on (mean= 6.7cm) than that off (mean= 11.9cm) prairie dog towns. This research indicates that cattle on the shortgrass steppe use prairie dog towns randomly and do not avoid them despite the shorter vegetation on prairie dog towns. However, cattle do not prefer to graze on prairie dog towns as bison (Bison bison) do on the mixed-grass prairie.
Open Access
Changing myoglobin's paradigm: characterizing the role between lipids and myoglobin expression
(Colorado State University. Libraries, 2014) Schlater, Amber E., author; Kanatous, Shane B., advisor; Florant, Gregory, committee member; Mykles, Donald, committee member; Bell, Christopher, committee member
Myoglobin (Mb) is a muscular heme protein generally localized to oxidative muscle, where it functions to store and transport oxygen, as well as scavenge nitric oxide and reactive oxygen species (ROS). While the former role of Mb in oxygen storage/transport is undisputed in diving mammals and other hypoxia-adapted species, this function appears to be far more ambiguous in terrestrial, non-hypoxia-adapted species. During endurance exercise, terrestrial mammals rely on erythrocytic oxygen to fuel aerobic metabolism in working muscle. Physiological changes associated with endurance training elicit responses that increase muscular blood flow and subsequent oxygen delivery. Intramuscular oxygen stores, alternatively, appear to bear little significance in sustaining aerobic metabolism during endurance exercise, as evident by the inability to appreciably release intra-muscular stored oxygen during normoxic exercise; yet, terrestrial endurance athletes who tend to have a higher reliance on lipid-fueled metabolism have more Mb than their sedentary counterparts. Accordingly, Mb's traditional functional paradigm pertaining to oxygen storage and transport does not appear to be fully applicable to terrestrial mammals in vivo. Here, a series of datasets are provided offering alternative paradigm, where increases in Mb expression are associated with increases in lipid supplementation. C2C12 cells cultured in normoxic and hypoxic (0.5% oxygen) environments show increased Mb when supplemented with a 5% lipid mixture compared to glucose controls. While Mb regulatory pathways have been shown to involve Ca2+ signaling pathways via calcineurin (CN), this lipid-induced Mb stimulation is not affiliated with an increase in CN expression, suggestive of a regulatory pathway for Mb independent of Ca2+. Moreover, lipid-induced Mb stimulation parallels oxidative stress marker augmentation concomitant with Mb augmentation. Addition of antioxidant to lipid-supplemented cells reverses Mb increases, and acute exposure to H2O2 during hypoxic differentiation showed an increase in Mb relative to control cells, collectively suggesting a Mb regulatory pathway through redox signaling. Furthermore, comparison of two commonly used Mb assay techniques revealed that normoxic lipid-induced Mb increases are nearly explicitly oxidized, thus bearing important functional implications on Mb increases consequent of lipid stimulation. In light of these novel data and in conjunction with the inability of terrestrial mammals to appreciably utilize Mb oxygen stores during exercise, an alternative paradigm for Mb is proposed. I propose that the role of Mb as an antioxidant defense during terrestrial exercise, which increases lipid-based aerobic metabolism and ROS production, is more relevant and applicable than a role relevant to storage and transport of oxygen.
Open Access
Charactarization of a nitrate responsive MYB transcription factor in Arabidopsis
(Colorado State University. Libraries, 2013) Stoerger, Vincent, author; Bush, Daniel, advisor; Bedinger, Patricia, committee member; Byrne, Patrick, committee member; Reddy, A. S. N., committee member
NRM1 (AT1g13300) is a gene that was initially uncovered in a microarray experiment where Arabidopsis was starved of nitrate and then re-fed varying concentrations of nitrate. NRM1 was one of a few genes that was up-regulated in all treatments. NRM1 has a single MYB like domain and a leucine-zipper like domain. We hypothesized that NRM1 is a transcription factor that plays a role in the plants response to nitrate availability.
Open Access
Characterization of a synthetic signal transduction system
(Colorado State University. Libraries, 2012) Albrecht, Tessa, author; Medford, June, advisor; Bush, Daniel, committee member; Pilon-Smits, Elizabeth, committee member; Leach, Jan, committee member
The Medford laboratory has developed a synthetic signal transduction system linking exogenous perception of a particular ligand to a transcriptional response. One application of this system is to produce plants that sense and respond to a specific ligand. The system was designed based on evolutionary conservation of histidine kinase signaling and uses bacterial components adapted to function in plants. The synthetic signaling system is responsive to extracellular ligand perception by a wild-type or modified ribose binding protein (RBP) scaffold. Upon ligand binding, RBP binds and activates a synthetic fusion histidine kinase made from the extracellular portion of the bacterial chemotactic receptor Trg and the cytoplasmic portion of the bacterial phosphate sensor PhoR. Activated Trg-PhoR transmits a phosphate signal to the bacterial response regulator PhoB. Upon phosphorylation PhoB translocates into the nucleus of a plant cell and activates transcription of the response gene(s). In addition to receiving a phosphate signal from Trg-PhoR, PhoB can be activated by exogenous cytokinin application suggesting that components of the cytokinin signaling pathway can interact with PhoB. Elimination or reduction of the interaction with cytokinin signaling components allows production of a more reliable signaling system. One goal of the following work was to reduce the interaction of PhoB with endogenous cytokinin signaling components. I attempted to identify a mutant form of PhoB that does not interact with cytokinin signaling components yet maintains function with the synthetic signaling system. I screened six different rationally selected PhoB mutants in plants for reduced response to exogenous cytokinin application. I concluded that a different approach will be needed to successfully reduce interaction with cytokinin signaling components. Another goal of this work was the identification cytokinin signaling components that interact with PhoB, possibly revealing a means to eliminate the interaction. I attempted to functionally express selected cytokinin signaling components in a bacterial testing system. After several failed cloning strategies, I conclude that the cytokinin sensor histidine kinase, AHK4, may be toxic and/or unstable in bacteria and expression of alternative genes will be needed to identify cytokinin signaling components that interact with PhoB. Additional work described here includes the independent testing of two computationally designed RBPs; one reported to bind the environmental pollutant methyl tert-butyl ether and the other reported to bind the explosive trinitrotoluene, for ligand dependent activation of the synthetic signaling system. These results show that the computationally designed RBPs do not function in a reliable manner and lead to the production of a detector plant using wild-type RBP to activate the synthetic signaling system that enables further analysis of the system components in plants.