Theses and Dissertations at Montana State University (MSU)
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Item Characteristics of whitebark pine (Pinus albicaulis) growth & defense in disturbance-prone, high-elevation, montane ecosystems of the northern Rocky Mountains(Montana State University - Bozeman, College of Letters & Science, 2022) Kichas, Nickolas Earl; Chairperson, Graduate Committee: David McWethy; This is a manuscript style paper that includes co-authored chapters.Whitebark pine (Pinus albicaulis) is a high-elevation conifer, recognized as a foundation species due to the numerous ecological benefits it provides in subalpine environments. In whitebark pine and other conifers, resin-based defenses have long been recognized as the primary mechanism by which trees respond to bark beetle attacks and several studies have linked resin duct properties to survivorship during periods of increased beetle activity. Utilizing a unique dataset of whitebark pine collected on the Flathead Indian Reservation in northwestern Montana, we set out to investigate the following research questions: (1) Are there differences in physiology (tree growth and resin duct anatomy) between trees that persisted through recent mountain pine beetle outbreaks and trees that died? (2) Does constitutive resin chemistry differ between whitebark and co-occurring lodgepole pine and are there relationships between tree growth, resin duct anatomy and resin chemistry? (3) Does competition influence constitutive resin chemistry in either whitebark or lodgepole pine? and (4) Is whitebark pine growth and/or resin duct anatomy constrained by warmer and/or regionally drier conditions? We found that whitebark pine trees that have persisted through recent stand-level disturbance produced fewer but larger resin duct structures with greater duct area compared to trees that died. We also detected important differences in the chemical composition of resin between whitebark and lodgepole pine that generally support field observations, whereby under endemic scenarios mountain pine beetle preferentially select lodgepole pine, while under outbreak scenarios, beetles successfully colonize whitebark pine trees. We found complex relationships between tree growth, resin duct anatomy and constitutive resin chemistry that present beetles with many permutations of resin-based defenses, while competition, particularly with Engelmann spruce (Picea engelmannii) can further influence constitutive resin chemistry. Lastly, we found that whitebark pine across our study sites are experiencing increased growth and defense under warmer and regionally drier conditions. Whitebark pine at our study sites exhibit differing strategies in the allocation of resources toward growth and defense. Our results support the idea that maintaining genetic variability promotes diverse response strategies to a complex array of biophysical stressors that might leave a species vulnerable to extinction across its range.Item Genomic, microbial, and linear relationships between residual feed intake, dry matter intake, average daily gain, and post weaning weights in range sheep(Montana State University - Bozeman, College of Agriculture, 2023) Schaub, Daniel James; Chairperson, Graduate Committee: Christian J. PosberghOver forty years, United States sheep producers have selectively bred range sheep for larger post-weaning weights using estimated breeding values. However, this has led to increased feed intake, prompting this study's objective to quantify the relationship between lamb growth estimated breeding values, feed intake, and feed efficiency. The study involved 40 Targhee and 40 Rambouillet ewe lambs weaned at four months and put on a GrowsafeTM feeding system for a 56-day experimental period, with ewes weighed weekly and at the start and end of the trial. Residual feed intake was calculated using body weights, average daily gain, and daily feed intake measurements. The ewe lambs' post-weaning weight estimated breeding value was associated to their phenotypic traits. These procedures were repeated when the same ewes were yearlings and at maintenance. The study found that while post-weaning weight estimated breeding values predicted dry matter intake (P<0.05), they lacked association with residual feed intake (P>0.05) in both experimental periods. However, the ewe's post-weaning weight estimated breeding value predicted mid-trial body weight in both periods (P<0.05). Thus, while selecting higher post- weaning weight animals may increase feed usage due to larger body size, it does not affect feed efficiency. Comparing the residual feed intake between the two experimental periods, growth residual feed intake measurements did not predict residual feed intake in maintenance. Therefore, caution should be used when utilizing residual feed intake from the growth period as an indicator of feed efficiency in maintenance. The study also examined rumen microbial profiles in sheep between the two experimental periods. Methanogenic archaea were relatively increased within the growth experimental period, where a pelleted diet was used, and cellulolytic bacteria were increased in the maintenance experimental period, where hay was provided. Since methanogens are associated with decreased feed efficiency, diet may play a role in changing a sheep's microbiome towards more feed efficient bacteria. Finally, a genome-wide association study found the Dmrt2 gene associated with DMI and RFI in maintenance. The Dmrt2 gene may regulate muscle and bone development in the early embryo, affecting feed efficiency in sheep later in life.Item Clodronate use in yearling, exercising horses on endocrine regulation of bone growth and development(Montana State University - Bozeman, College of Agriculture, 2023) Conrad, Matthew Benjamin; Chairperson, Graduate Committee: Amanda N. BradberyExtra-label bisphosphonate use in juvenile horses is widespread despite limited scientific understanding of biological and welfare impacts on skeletally immature, exercising horses. The objective of this study was to determine the effects of clodronate on endocrine regulation of bone growth, hypothesizing there would be no effect. To test this, 32 Quarter Horses were stratified by age (500 + or - 13 d of age), BW (336 + or - 26 kg), sex (n = 16 males, n = 16 females), and initial bone optical density by aluminum equivalence into four treatment groups for a 168-d trial. The experimental period was divided into two phases mimicking sales preparation (Phase I: d 0-83) and early performance training (Phase II: d 84-168). Investigators were blinded, and all horses received iso-volumetric intramuscular injections of either 1.8 mg/kg BW clodronate disodium (OSPHOS®) or saline (placebo) on d 0, 42, 84, and 126. Treatments consisted of control (CON; n = 8), single-dose (1X; n = 8; d 84), two-dose (2X; n = 8; d 0, 84), and four-dose groups (4X; n = 8; d 0, 42, 84, 126). Physical measurements including BW, wither height (WH), hip height (HH), body length (BL), and heart girth (HG) circumference were recorded every 42 d from d 0. Serum samples were collected every 42 d prior to treatment administration to quantify growth hormone (GH), calcitonin (CT), parathyroid hormone (PTH), and ionized calcium. Data were analyzed using PROC MIXED of SAS. All physical growth parameters increased (P < or = 0.01) and growth hormone tended to decrease over time (P = 0.09) following normal growth patterns. A treatment time interaction was observed for PTH (P = 0.05) where concentrations increased on d 84 in 4X and on d 126 in 2x following the second administration of clodronate while 1X and CON remained unchanged. Ionized Ca decreased over time (P < 0.01) with no effect on CT concentrations (P > 0.33). These results suggest that while administration of bisphosphonate has no effect on growth morphometrics, its repetitive use alters PTH concentrations after two doses.Item Distribution, phenology, growth, and overwinter mortality of age-0 smallmouth bass in the Yellowstone River, with implications for upstream range expansion(Montana State University - Bozeman, College of Letters & Science, 2020) Voss, Nicholas Sheridan; Chairperson, Graduate Committee: Alexander V. Zale; Robert Al-Chokhachy, Adam J. Sepulveda, Christine E. Verhille, Michael P. Ruggles and Alexander V. Zale were co-authors of the article, 'Distribution, phenology, growth, and overwinter mortality of age-0 smallmouth bass in the Yellowstone River, with implications for upstream range expansion' submitted to the journal 'Transactions of the American Fisheries Society' which is contained within this thesis.Non-native fish introductions are a leading threat to freshwater biodiversity, and accurate assessments of future impact are often hindered by the challenge of anticipating future range expansion. Successful introductions of non-native Smallmouth Bass Micropterus dolomieu have occurred globally and often exhibit secondary spread to upstream habitat. This has occurred in the Yellowstone River, Montana (USA). Observations of adults in socio-economically valuable trout habitat have highlighted a need to better understand the controls on the upstream distribution of Smallmouth Bass in this system, particularly the influence of cold upstream climates on first-year growth and size-selective overwinter mortality (a potential life history bottleneck at northern latitudes). We documented the phenology, growth, and survival of age-0 Smallmouth Bass in relation to water temperature between the uppermost distribution of adults, and downstream regions where they are abundant. Successful reproduction (i.e., age-0 presence) was rare or absent throughout the uppermost 150 km of the upstream distribution of adults, suggesting that something currently prevents or discourages successful reproduction farther upstream. Surprisingly, the mean late-autumn body size of age-0 Smallmouth Bass did not differ significantly among the uppermost 200 km of their distribution, despite upstream declines in ambient water temperature. Although water temperature was a key attribute affecting age-0 growth, upstream shifts towards earlier hatching mediated the expected negative effect of colder upstream climates. Furthermore, surveys of overwinter survivors and simulations of age-0 starvation mortality indicated that age-0 individuals at the upstream extent of their distribution successfully recruited to the age-1 year-class in four consecutive years. Taken together, our results suggest that Smallmouth Bass have not yet reached the thermal limit of their upstream distribution, and that first-year growth, survival, and consequent spread by this non-native predator are probably driven by the complex interactions of spawn timing and ambient thermal and hydrologic regimes in the Yellowstone River.Item Evaluation of nitorgen and carbon supplementation strategies for optimizing biomass generation during cultivation of Chlorella sorokiniana, strain SLA-04(Montana State University - Bozeman, College of Engineering, 2021) Jackson, Matthew Clifford; Chairperson, Graduate Committee: Catherine KirklandAlgal cultivation requires significant nitrogen and carbon inputs, which are expensive and can offset benefits associated with biofuel production. This research investigates growth of an alkali-tolerant Chlorella sorokiniana, strain SLA-04, using different nitrogen and carbon regimes to improve physiological knowledge of this novel organism, and improve biomass production and resource demand. Nitrate, ammonium, and urea were used efficiently by SLA-04, however pH changes during utilization of nitrate and ammonium impacted inorganic carbon availability (species and concentration). Generation of OH- during use of nitrate increased pH, increasing mass transfer of CO 2 into solution and increasing the ratio of HCO 3-/CO 2. Ammonium utilization resulted in proton generation, lowering pH and inhibiting growth. When bicarbonate, rather than CO 2, was provided, productivity improved for the urea and mixed nitrogen conditions. This likely resulted from upregulation of genes related to nitrogen and carbon assimilation in the presence of bicarbonate, however Na + cotransport with urea and nitrate is required in some organisms. It is possible that Na + was insufficient when CO 2 was provided, but not in conditions with bicarbonate since it was added as NaHCO 3-. The impact of Na +, as well as other ions, on nitrogen and carbon utilization is not well understood, but it may alter gene regulation. Bicarbonate and CO 2 both promoted increased growth relative to cultures without inorganic carbon supplementation. The highest productivities were observed when carbon supplementation, either as continued CO 2 augmentation to the air sparge or as a 50mM bicarbonate amendment, was provided during nitrogen deplete growth. Glucose availability improved productivity for conditions without CO 2 supplementation. The use of urea or a combination of nitrogen sources with bicarbonate, instead of CO 2, was promising due to (a) the low cost of urea, relative to the other nitrogen sources; (b) the potential for using wastewater containing a mix of nitrogen sources; and (c) the low cost and easy transport of bicarbonate. Future research should evaluate changes in SLA-04 gene expression resulting from the supply of different nutrients, including nitrogen and carbon sources, as well as other ions essential for growth.Item Comparing network models of gap gene interaction during Drosophila melanogaster development(Montana State University - Bozeman, College of Letters & Science, 2021) Andreas, Elizabeth Anne; Chairperson, Graduate Committee: Tomas GedeonEarly development of Drosophila melanogaster (fruit fly) facilitated by the gap gene network has been shown to be incredibly robust, and the same patterns emerge even when the process is seriously disrupted. In this thesis we plan to investigate this robustness using a previously developed computational framework called Dynamic Signatures Generated by Regulatory Networks (DSGRN). The principal result of this research has been in extending DSGRN to study how tissue-scale behavior arises from network behavior in individual cells, such as gap gene expression along the anterior-posterior (A-P) axis of the Drosophila embryo. Essentially, we extend DSGRN to study cellular systems where each cell contains the same network structure but operates under a parameter regime that changes continuously from cell to cell. We then use this extension to study the robustness of two different models of the gap gene network by looking at the number of paths in each network that can produce the observed gap gene expression. While we found that both networks are capable or replicating the data, we hypothesize that one network is a better fit than the other. This is significant in two ways; finding paths shows us that the spatial data can be replicated using a single network with different parameters along the A-P axis, and that we may be able to use this extension of DSGRN to rank network models.Item Understanding physiological adaptations, metabolic potential and ecology in a novel photoautotrophic alga for biofuel production(Montana State University - Bozeman, College of Letters & Science, 2019) Corredor Arias, Luisa Fernanda; Chairperson, Graduate Committee: Matthew Fields; Elliot B. Barnhart, Al Parker, Robin Gerlach and Matthew W. Fields were co-authors of the article, 'Impact of temperature, nitrate concentration, PH and bicarbonate addition on biomass and lipid accumulation of a sporulating green alga' which is contained within this dissertation.; Thiru Ramaraj, Huyen Bui, Mensur Dlakic, Robin Gerlach and Matthew W. Fields were co-authors of the article, 'Genomic insights into a sporulating, non-motile, oligotrophic green microalga (PW95)' which is contained within this dissertation.; Huyen Bui, Thiru Ramaraj, Robin Gerlach and Matthew W. Fields were co-authors of the article, 'Transcriptomic profiling of Chlamydomonas-like PW95 cultivated in coal bed methane production water with the native microbial community' which is contained within this dissertation.; Anna J. Zelaya, Robin Gerlach and Matthew W. Fields were co-authors of the article, 'Associations between sympatric bacterial groups and a novel green alga cultivated in coal bed methane production water' which is contained within this dissertation.Commercial implementation of microalgal biomass as bio-oil/chemical feedstocks has been difficult to achieve, and challenges include water/nutrient sources, CO 2 delivery, and community dynamics of mixed cultures. We employed an integrated approach to the study of microalgal production systems to advance towards sustainable implementation of industrial microalgal biofuel production using a native alga (Chlamydomonas-like alga, PW95) isolated from Coal Bed Methane (CBM) production water. Our approach was based on the evaluation of PW95 physiological responses to combinations of growth constraints, the determination of its genomic and functional potential, phylogenetic relations and the implementation of an ecosystem view to algal biomass production. PW95 growth and lipid accumulation (biofuel potential) were ascertained in standardized media and CBM water through the evaluation of mixed effects of temperatures, nitrate levels, pH, and bicarbonate to elucidate interactions between multiple environmental variables and nutritional levels. The biofuel potential of PW95 ranges between 20-32% depending on culture conditions and our results suggest an important interaction between low nitrate levels, high temperature, and elevated pH for trade-offs between biomass and lipid production in the alga. Whole genome sequence was employed to predict biological and metabolic capacity in PW95, and the expression of these capabilities during growth in CBM water with the native microbial consortia was evaluated using RNA sequencing. genome determination and assembly resulted in a draft genome size of 92 Mbp with 14,000 genes predicted and 402 pathways mapped in the KEGG database. The gene complement of PW95 provided a glance into life in an oligotrophic environment (CBM water) and evidence of essential metabolic pathways for cell growth, survival and maintenance, also relevant for cultivation and value-added products generation. Microbial composition and shifts during growth were identified, as well as the algal phycosome. During growth in CBM water, PW95 appeared to be supported by a native microbial consortium and differential expression analysis showed basic metabolic functions and adaptive physiological responses. Our findings build on previous knowledge for improved algal culturing for biomass and industry-valued products while exploring the biology of an organism with relevant impact in energy and water resource management.Item Improving osseointegration of PEEK through surface textures(Montana State University - Bozeman, College of Engineering, 2019) Scott, Renn Patrick; Chairperson, Graduate Committee: Cecily RyanPEEK (Polyetheretherketone) is one of the most promising alternatives to titanium in cortical bone implants due to being biologically inert and having an elastic modulus similar to that of bone. It also has favorable reactions conducive to common medical imaging methods such as X-ray and magnetic resonance imaging (MRI) as compared to commonly used metals such as titanium and stainless steel. However PEEK is not inherently osseoconductive, leading to longer healing times and a greater chance of infection. Many different methods exist for improving osteoblast growth, such as the addition of bio-active materials like hydroxyapatite. Manipulating the surface texture of PEEK could provide better environments for cells to attach and can be used as another layer with other techniques, making the tissue interface more robust. The main objective of this project is to observe cell adhesion to a textured surface to identify cell preference for surface geometry as a first step to improve full integration of non-resorbable implants into bone tissue. The methods explored were also chosen for their repeatability, reliability and lack of chemical modification compared to other successful surface modulation techniques. The surface textures were embossed into PEEK using micro-etched aluminum molds. Textures vary in their shape, spacing, size, depth and surface convexity/concavity. The cell adhesion was recorded through fluorescent confocal microscopy and the cell-substrate interaction was observed under electron microscopy. The results were that 25 micron and 10 micron features discouraged cell adhesion while 325 micron and 120 micron features encouraged cell adhesion with pillars performing better than holes. The best feature was the 325x40 micron square pillars. With a cell volume to surface area ratio of 5.13, a live cell count of 276.5, a dead cell count of 9.00, and a non-dimensional distance to feature of 0.67.Item Spatiotemporal mapping of oxygen in model porous media biofilms using 19 F magnetic resonance oximetry(Montana State University - Bozeman, College of Engineering, 2019) Simkins, Jeffrey William; Chairperson, Graduate Committee: Philip S. Stewart and Joseph D. Seymour (co-chair); Philip S. Stewart and Joseph D. Seymour were co-authors of the article, 'Spatiotemporal mapping of oxygen in a microbially-impacted packed bed using 19 F nuclear magnetic resonance oximetry' in the journal 'The journal of magnetic resonance' which is contained within this dissertation.; Philip S. Stewart, Sarah L. Codd and Joseph D. Seymour were co-authors of the article, 'Non-invasive imaging of oxygen concentration in a complex in vitro biofilm infection model using 19 F MRI: persistence of an oxygen sink despite prolonged antibiotic therapy' submitted to the journal 'Magnetic resonance in medicine' which is contained within this dissertation.; Philip S. Stewart and Joseph D. Seymour were co-authors of the article, 'Microbial growth rates and local external mass transfer resistance in a porous bed biofilm system measured by 19 F magnetic resonance imaging of structure, oxygen concentration, and flow velocity' submitted to the journal 'Biotechnology and bioengineering' which is contained within this dissertation.Biofilms, microbial aggregates anchored to a surface using a sticky matrix of metabolic products called extracellular polymeric substances (EPS), are the dominant form of bacterial life and are widespread in nature, from glaciers to hot springs. The transition from the planktonic state to a biofilm is associated with striking changes to microbial phenotype which confer unique, biofilm-specific properties to resident cells that have important implications for medicine, industry, and environmental study. Many of these properties are caused in large part by oxygen transport limitation, which arises due to restriction of fluid flow in cell aggregates and consumption of oxygen for respiration. The balance of reactive and diffusive processes establishes strong spatial gradients in oxygen concentration which lead to profound spatial heterogeneity in bacterial species composition, growth yield, antimicrobial susceptibility, and reaction kinetics, among other traits. However, despite the importance of oxygen gradients in a host of highly-relevant biofilm phenomena, quantification of oxygen profiles in biofilms is difficult, both in the field and the lab, with the gold standard of measurement, the microelectrode, having significant limitations. 19 F Nuclear Magnetic Resonance (NMR) oximetry, a magnetic resonance-based technique for oxygen quantification that has been used to characterize oxygen usage in blood tissues and tumors, exploits the linear dependence of spin-lattice relaxation rate R 1 on local oxygen partial pressure for fluorine nuclei in perfluorocarbon (PFC) phases. In the current work, we apply 19 F NMR oximetry to a model packed bed biofilm system to generate novel insights into microbial oxygen usage and to introduce a complimentary oximetry tool for biofilm experimenters. We develop methodology for the introduction and fixation of a fluorinated oxygen sensor to facilitate long-term oxygen monitoring. We use 19 F oxygen distribution measurements in compliment to traditional NMR methods to correlate fluid flow with growth rate, generate spatial maps of oxygen utilization rate, identify differences in oxygen utilization behavior between different species, characterize infection persistence during antibiotic therapy, mathematically model macroscale oxygen sink development, and quantify local mass transfer phenomena.Item Description of the reproductive structure, size, growth, and condition of hatchery-origin white sturgeon in the lower Columbia River, British Columbia, Canada(Montana State University - Bozeman, College of Letters & Science, 2020) Maskill, Paige Anne Camas; Chairperson, Graduate Committee: Christopher S. Guy; James A. Crossman, Christopher S. Guy and Molly A. H. Webb were co-authors of the article, 'Describing the reproductive structure of the hatchery-origin population of white sturgeon in the lower Columbia River, British Columbia, Canada' which is contained within this thesis.Conservation aquaculture has been successful in preventing extirpation of endangered white sturgeon Acipenser transmontanus in the lower Columbia River (LCR), Canada. The goals of this project were to 1) determine if the hatchery-origin (HO) population has reached puberty as many fish are of an age and body size to begin contributing to spawning and 2) assess the size, absolute growth rate (AGR), and relative condition factor (Kn) of the population. The reproductive structure (sex and stage of maturity) of the HO population was assessed using multiple tools (gonadal biopsy analyzed histologically, ultrasound, endoscopy, and plasma sex steroids). The accuracy of each tool was determined by comparing the assigned sex to the true sex determined by histological analysis of a gonadal biopsy. A subobjective was to determine whether gonadal development was homogenous (assessed in fish captured in LCR and Lake Roosevelt, WA, USA). All fish assessed were pre-meiotic males (n=158) or pre-vitellogenic females (n=174) and had yet to reach puberty. Endoscopy and gonadal biopsy analyzed histologically were the most accurate tools with accuracies > 97%. Gonadal development was homogenous in females but 38% of the males exhibited non-homogenous testicular development, with a few precocious cysts that contained germ cells in an advanced stage of development compared to the predominant phase seen throughout the biopsy. The size, AGR, and Kn were compared among sex, age, and river zone. While the mean Kn indicated that the HO white sturgeon were in average condition relative to all HO fish within the LCR, they were lower in condition compared to the full transboundary population (Canada and USA). This reflects a difference in the environmental or physiological conditions of the fish in the LCR and those captured throughout the transboundary reach. Results of this work will be important to determine the reproductive dynamics of the hatchery-origin population over time (e.g., age and size at puberty, sex ratio), and specifically when these fish will begin spawning in the wild. Additionally, this information will help to develop other standardized monitoring programs which can track the reproductive structure of a population over time using tools with known accuracy rates.