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Description
The termite Coptotermes gestroi is a small subterranean termite originating from Southeast Asia. The hindgut of C. gestroi contains five distinct species of parabasalid: Pseudotrichonympha leei, two undescribed species from the genus Holomastigotoides, and two undescribed species from the genus Cononympha. This study investigates the protist symbionts in C. gestroi and the relationship between their relative abundance as inferred by Illumina sequence reads and the directly observed abundances for each protist genus. Illumina amplicon sequencing as a means of DNA analysis is a proven method for identification and diversity analysis, although the specific ratios of sequence reads to cell abundance in protists is not well known. In this study, protist communities were observed under light microscopy; cells were counted under hemocytometer and characterized at the molecular level using Illumina amplicon sequencing. When comparing sequence read abundances to cell abundances, some general trends were found in both analysis methods. Cononmypha repeatedly formed the majority of the community, while Holomastigotoides and Pseudotrichonympha were responsible for a smaller yet similar portion of the population. Cell counts and sequence reads were also compared using an assumed linear model, with R2 values generated to quantify the relationship between both. The results suggest that Illumina sequencing can be used to obtain rough estimates of community diversity, but the high variability within the data suggest that the read abundances should be treated with caution.
ContributorsAvilucea, Erin L. (Author) / Gile, Gillian (Thesis director) / DeMartini, Francesca (Committee member) / Taerum, Stephen Joshua (Committee member) / School of Mathematical and Natural Sciences (Contributor) / Barrett, The Honors College (Contributor)
Created2018-05
Description
Antibiotic resistance in the modern era has reached near-epidemic levels, resulting in much more difficult treatment of previously well-managed pathogens. Previous understandings of how antibiotic resistance emerges failed to account for the function of the environment. Over the past 15 years, new research has provided a link between the environmental and clinical spheres of antibiotic use. This data suggests that environmental bacteria, particularly those found in livestock farming ecosystems, may significantly contribute to the overall flow of antibiotic resistance genes into human populations. The main force behind this is the utilization of antibiotics as growth promoters in animal feed supplements, seeding individual animals and their surroundings with low doses of antibiotics. Notable increases in resistance have been observed within areas that utilize these supplements, as well as in connected but unrelated systems. Waste management strategies are poorly implemented, leading to the dispersal of contaminated runoff into groundwater and riverine environments. Furthermore, existing waste processing is limited in efficacy, often releasing large amounts of unprocessed antibiotics as well as a concentrated population of resistant bacteria. Within these resistant populations, horizontal gene transfer has emerged as a vehicle for the distribution of resistance genes into other populations of bacteria. Due to the prevalence of these transfer events, a new role for the environment as a reservoir and incubator of resistance genes is proposed. Current strategies for managing the spread of antibiotic resistance are woefully inadequate, and the continued emergence of new resistance mechanisms due to negligence highlights the need for global, multidisciplinary solutions. To corral the spread of antibiotic resistance, a system is proposed that utilizes metagenomic monitoring and the enforcement of core global policies to slow the advance of resistance while waiting for novel treatment strategies to bear fruit.
ContributorsHrkal, Jacob (Author) / Gile, Gillian (Thesis director) / Shi, Yixin (Committee member) / Sarno, Analissa (Committee member) / School of Life Sciences (Contributor) / Barrett, The Honors College (Contributor)
Created2016-05
Description
Hundreds of thousands of people die annually from malaria; a protozoan of the genus Plasmodium is responsible for this mortality. The Plasmodium parasite undergoes several life stages within the mosquito vector, the transition between which require passage across the lumen of the mosquito midgut. It has been observed that in about 15% of parasites that develop ookinetes in the mosquito abdomen, sporozoites never develop in the salivary glands, indicating that passage across the midgut lumen is a significant barrier in parasite development (Gamage-Mendis et al., 1993). We aim to investigate a possible correlation between passage through the midgut lumen and drug-resistance trends in Plasmodium falciparum parasites. This study contains a total of 1024 Anopheles mosquitoes: 187 Anopheles gambiae and 837 Anopheles funestus samples collected in high malaria transmission areas of Mozambique between March and June of 2016. Sanger sequencing will be used to determine the prevalence of known resistance alleles for anti-malarial drugs: chloroquine resistance transporter (pfcrt), multidrug resistance (pfmdr1) gene, dihydropteroate synthase (pfdhps) and dihydrofolate reductase (pfdhfr). We compare prevalence of resistance between abdomen and head/thorax in order to determine whether drug resistant parasites are disproportionately hindered during their passage through the midgut lumen. A statistically significant difference between resistance alleles in the two studied body sections supports the efficacy of new anti-malarial gene surveillance strategies in areas of high malaria transmission.
ContributorsPhillips, Keeley Isabella (Author) / Huijben, Silvie (Thesis director) / Gile, Gillian (Committee member) / Young, Steven (Committee member) / School of Life Sciences (Contributor) / Barrett, The Honors College (Contributor)
Created2021-05
Description
The 18S ribosomal RNA gene is ubiquitous across eukaryotes as it encodes the RNA component of the ribosomal small subunit. It is the most commonly used marker in molecular studies of unicellular eukaryotes (protists) due to its species specificity and high copy number in the protist genome. Recent studies have revealed the widespread occurrence of intragenomic (intra-individual) polymorphism in many protists, an understudied phenomenon which contradicts the assumed homogeneity of the 18S throughout an individual genome. This thesis quantifies and analyzes the level of intragenomic and intraspecific 18S sequence variability in three Trichonympha species (T. campanula, T. collaris, T. postcylindrica) from Zootermopsis termites. Single-cell DNA extractions, PCR, cloning, and sequencing were performed to obtain 18S rRNA sequence reads, which were then analyzed to determine levels of sequence divergence among individuals and among species. Intragenomic variability was encountered in all three species. However, excluding singleton mutations, sequence divergence was less than 1% in 53 of the 56 compared individuals. T. collaris exhibited the most substantial intragenomic variability, with sequence divergence ranging from 0 to 3.4%. Further studies with more clones per cell are needed to elucidate the true extent of intragenomic variability in Trichonympha.
ContributorsBobbett, Bradley (Author) / Gile, Gillian (Thesis director) / Liebig, Juergen (Committee member) / School of International Letters and Cultures (Contributor) / School of Life Sciences (Contributor) / Barrett, The Honors College (Contributor)
Created2020-05
Description
Predatory bacteria are a guild of heterotrophs that feed directly on other living bacteria. They belong to several bacterial lineages that evolved this mode of life independently and occur in many microbiomes and environments. Current knowledge of predatory bacteria is based on culture studies and simple detection in natural systems. The ecological consequences of their activity, unlike those of other populational loss factors like viral infection or grazing by protists, are yet to be assessed. During large-scale cultivation of biological soil crusts intended for arid soil rehabilitation, episodes of catastrophic failure were observed in cyanobacterial growth that could be ascribed to the action of an unknown predatory bacterium using bioassays. This predatory bacterium was also present in natural biocrust communities, where it formed clearings (plaques) up to 9 cm in diameter that were visible to the naked eye. Enrichment cultivation and purification by cell-sorting were used to obtain co-cultures of the predator with its cyanobacterial prey, as well as to identify and characterize it genomically, physiologically and ultrastructurally. A Bacteroidetes bacterium, unrelated to any known isolate at the family level, it is endobiotic, non-motile, obligately predatory, displays a complex life cycle and very unusual ultrastructure. Extracellular propagules are small (0.8-1.0 µm) Gram-negative cocci with internal two-membrane-bound compartmentalization. These gain entry to the prey likely using a suite of hydrolytic enzymes, localizing to the cyanobacterial cytoplasm, where growth begins into non-compartmentalized pseudofilaments that undergo secretion of vesicles and simultaneous multiple division to yield new propagules. I formally describe it as Candidatus Cyanoraptor togatus, hereafter Cyanoraptor. Its prey range is restricted to biocrust-forming, filamentous, non-heterocystous, gliding, bundle-making cyanobacteria. Molecular meta-analyses showed its worldwide distribution in biocrusts. Biogeochemical analyses of Cyanoraptor plaques revealed that it causes a complete loss of primary productivity, and significant decreases in other biocrusts properties such as water-retention and dust-trapping capacity. Extensive field surveys in the US Southwest revealed its ubiquity and its dispersal-limited, aggregated spatial distribution and incidence. Overall, its activity reduces biocrust productivity by 10% at the ecosystem scale. My research points to predatory bacteria as a significant, but overlooked, ecological force in shaping soil microbiomes.
ContributorsBethany Rakes, Julie Ann (Author) / Garcia-Pichel, Ferran (Thesis advisor) / Gile, Gillian (Committee member) / Cao, Huansheng (Committee member) / Jacobs, Bertram (Committee member) / Arizona State University (Publisher)
Created2022
Description
Lophomonas is a genus of flagellated parabasalids that exist as commensal symbionts in the hindguts of a variety of pest cockroaches. The genus contains two species: Lophomonas blattarum and Lophomonas striata. The two species differ by way of bacterial ectosymbionts that attach to the outside of L. striata, giving rise to a striated and spindle-shaped appearance. As the attachment of bacterial symbionts prohibits L. striata from taking up large food particles in the same manner as L. blattarum, it is likely the two species differ in which metabolic genes they possess. Here, a comparison of transcriptomes between the two Lophomonas species show slight differences between the species. Metagenomic analysis of L. striata also presents the possibility of L. striata ectosymbionts as belonging to the genus Parabacteroides.
ContributorsNguyen, Leann (Author) / Gile, Gillian (Thesis advisor) / Dada, Nsa (Committee member) / Wideman, Jeremy (Committee member) / Arizona State University (Publisher)
Created2023
Description
Biocrusts are microbial communities that inhabit arid soil surfaces, providing essential services to dryland ecosystems. A paradoxical filamentous cyanobacterium, Microcoleus vaginatus, resides within the biocrust. While is often pioneers the colonization of bare, nutrient-poor desert soils worldwide, it cannot fix dinitrogen. In nature, M. vaginatus coexists with a unique microbial community, a “cyanosphere”, that is characterized by a high abundance of diazotrophic heterotrophs. This suggests mutualistic relationships wherein nutrients are traded between phototrophs and heterotrophs. To explore these relationships, I performed targeted, pedigreed isolation of cyanosphere members and used co-cultivation to recreate the mutualism in culture. Results showed that, in the absence of fixed nitrogen, M. vaginatus grew well when co-cultured with cyanosphere diazotrophs, but only poorly or not at all when alone or with non-cyanosphere diazotrophs. In agreement with this, the experimental provision of nitrogen to natural populations resulted in a loss of diazotrophs from the cyanosphere compared to controls, but the addition of phosphorus did not. Additionally, the convergence of M. vaginatus trichomes into large bundles held by a common sheath was elicited in culture by the addition of cyanosphere diazotrophs, pointing to a role of cyanobacterial motility responses in the development of mutualistic interactions. I then demonstrated that the tendency of M. vaginatus to stay within bundles and close to the sheath-dwelling cyanosphere was dependent on the cyanosphere population size. This effect was likely mediated by glutamate that acted as a signaling molecule rather than as a N source and impacted the gliding speed and negative chemophobic responses on the cyanobacterium. Glutamate seems to be used as a cue to spatially optimize cyanobacterium-cyanosphere mutualistic exchanges. My findings have potential practical applications in restoration ecology, which I further pursued experimentally. Co-inoculation of soil with cyanosphere diazotrophs resulted in swifter development of biocrusts over inoculation with the cyanobacterium only. Further, their addition to disturbed native soils containing traces of cyanobacteria sufficed for the formation of cohesive biocrusts without cyanobacterial inoculation. The inclusion of such “biocrust probiotics” in biocrust restoration is recommended. Overall, this body of work elucidates the hitherto unknown role of beneficial heterotrophic bacteria in the initial formation and development of biocrusts.
ContributorsNelson, Corey (Author) / Garcia-Pichel, Ferran (Thesis advisor) / Penton, C. Ryan (Committee member) / Gile, Gillian (Committee member) / Bean, Heather (Committee member) / Arizona State University (Publisher)
Created2021