Vector competence of mosquitoes has been linked to the conditions in which the larvae mature to adults. The microbial community obtained from the rearing environment is suspected to be one key factor in this interplay. A better understanding of how the rearing environment affects the gut microbiome and Anopheles-Plasmodium interactions could be useful for understanding observed lab vs. field differences in Plasmodium biology and help drive future control efforts. Currently there is a lack of research done on the differences between lab strain mosquitoes and their rearing environments and how lab mosquitoes differ from wild type mosquitoes. Bridging this gap and studying how rearing habitats change gut microbiomes is critical for optimizing the lab-rearing environment. This thesis focuses on the effects larval rearing has on microbiome establishment and innate immune responses in the common malaria mosquito, Anopheles quadrimaculatus.

There is increasing evidence indicating that exposure to air pollutants may be associated with the onset of several respiratory diseases such as allergic airway disease and chronic obstructive pulmonary disorder (COPD). Many lung diseases demonstrate an outgrowth of pathogenic bacteria belonging to the Proteobacteria phylum, and the incidence of occurrence of these diseases is higher in heavily polluted regions. Within the human body, the lungs are among the first to be exposed to the harmful effects of inhaled pollutants and microbes. Research in the past few decades have expounded on the air-pollution-induced local and systemic inflammatory responses, but the involvement of the lung microbial communities has not yet been well-characterized. Lungs were historically considered to be sterile, but recent advances have demonstrated that the lower respiratory tract is replete with a wide variety of microorganisms - both in health and disease. Recent studies show that these lung microbes may play a significant role in modulating the immune environment by inducing IgA and mucus production. Air pollutants have previously been shown to alter intestinal bacterial populations that increase susceptibility to inflammatory diseases; however, to date, the effects of traffic-generated air pollutants on the resident microbial communities on the lungs have not been explored. The microbiome is influenced by several factors, including diet and environmental exposures. A large percentage of the Western world population consumes a high-fat (HF) diet which has resulted in the epidemic of obesity. Consumption of an HF diet has been shown to alter the intestinal microflora and increase baseline inflammation. We aimed to understand whether diet might also contribute to the alteration of the commensal lung microbiome, either alone or related to exposure. Thus, we investigated the hypothesis that exposure to air pollutants can alter the commensal lung microbiota, thereby promoting alterations in the lung's immune and inflammatory responses; in addition to determining whether these outcomes are exacerbated by a high fat-diet. We performed two studies with exposures to different components of air pollutant mixtures on C57Bl/6 mice placed on either a control (LF) diet or a high-fat (HF) diet. Our first exposure study was performed on C57Bl/6 mice with a mixture of gasoline and diesel engine emissions (ME: 30 µg PM/m3 gasoline engine emissions + 70 µg PM/m3 diesel engine emissions) or filtered air (FA) for 6h/d, 7 d/wk for 30 days. The ME study investigated the alterations in immunoglobulin A (IgA), IgG and IgM, and lung microbiota abundance and diversity. Our results revealed ME exposures alongside the HF diet causes a decrease in IgA and IgG when compared to FA controls, thereby decreasing airway barrier protection. This was accompanied by the expansion of bacteria within the Proteobacteria phylum and a decrease in the overall bacterial diversity and richness in the exposed vs. control groups. In our second study, we exposed C57Bl/6 mice to only the diesel exhaust particle component (35µg DEP, suspended in 35µl 0.9% sterile saline) or sterile saline only (control) twice a week for 30 days. We investigated immunoglobulin profiles by ELISA that revealed a significant increase in IgA and IgG in response to DEP. We also observed an increase in inflammatory tumor necrosis factor (TNF) - α, Interleukin (IL) -10, Toll-like receptors (TLR) - 2,4, nuclear factor kappa B (NF-κB) histologically and by RT-qPCR. Mucus production and collagen deposition within the lungs were also significantly elevated with DEP exposures. Microbial abundance determined quantitatively from the bronchoalveolar lavage fluid (BALF) by qPCR revealed an expansion of bacteria belonging to the Proteobacteria phylum in the DEP exposed groups on the HF diet. We also observed an increase in reactive oxygen and nitrogen species (ROS-RNS) products (nitrates), within the groups that revealed an expansion of Proteobacteria. These observations are most likely due to the unique metabolic capabilities of Proteobacteria to proliferate in inflammatory environments with excess nitrates. We assessed if treatments with probiotics could attenuate the DEP-induced inflammation by supplementing a separate group of study animals on the HF diet with 0.3 g/day of Winclove Ecologic® Barrier probiotics in their drinking water throughout the study. With probiotic treatments, we observed a significant decrease in ROS-RNS that was accompanied by complete elimination of Proteobacteria suggesting that in the absence of nitrates, the expansion of Proteobacteria is curbed effectively. We also observed a decrease in proinflammatory TNF-α and collagen deposition with probiotic treatments, and an increase in IgA levels within the BALF, suggesting that probiotics aid in balancing proinflammatory responses and enhance beneficial immune responses to efficiently mediate the DEP-induced inflammation. Both studies showed that air pollutants alter the immune defenses and contribute to lung microbial alterations with an expansion of Proteobacteria. The immunoglobulin profiles discordant between the two studies can be explained by the route and/or duration and composition of air pollutant exposure. Collectively these studies suggest that exposure to air pollutants alter immune responses and/or increase the availability of inflammatory by-products within the lungs that can enable the selective outgrowth of pathogenic bacteria. The observed detrimental outcomes are further exacerbated when coupled with the consumption of an HF diet. Importantly, these results may shed light on the missing link between air pollution-induced inflammation and bacterial expansion and also point to therapeutic alternatives to curb bacterial outgrowth in lung disease exacerbations observed in patient populations living and/or working in heavily polluted regions.

Up to 96% of angiosperms are dependent on animal pollinators for successful reproduction thus providing a key ecosystem service. Recent studies however, have indicated that micro-organisms inhabiting floral nectar can influence interactions between plants and their pollinators with consequences for plant fitness. Specifically, floral nectar provides suitable habitat for fungal and bacterial species i.e., the nectar microbiome (NMB), which can alter the amount and quality of nectar, floral volatile composition and impact pollinator preference and plant reproductive success. While the extent and consequences of these interactions are well understood for one or few plant species, community-level studies on the structure, drivers and fitness consequences of interactions between plants and nectar inhabiting microorganism are scarce. Community-level of studies of the drivers and consequences of interactions between plants and nectar microorganisms can help advance our understanding of the mechanisms that mediate the structure of plant-pollinator networks and mediate plant community assembly. In this study, we describe the structure of a plant-nectar microbiome network in a serpentine plant community in Northern California. We further evaluate how the structural properties of such network are mediated by the abundance and diversity of floral visitors and its effects on plant reproductive success. Nectar samples were collected from 15 plant species and cultured on agar plates until isolated. Fungal ITS and bacterial 16s DNA sequences were used for identification by Sanger sequencing. We further explored the effects pollinator visitation on of plant-microbiome network properties and the effects of the latter on pollen deposition and pollen tube production. Plant-nectar microbiome network was composed of 52 fungal and 27 bacterial morphospecies (79 total cultured CFU). Filamentous fungi (i.e. molds) comprise 80% of all fungal colonies. Network analysis also revealed network connectance and specialization were lower (0.07 and 0.39 respectively) than reported for plant-pollinator networks in the same community. On average, individual plants have 2.32 links with modularity and nestedness values of 0.36 and 0.32, respectively, which are comparable to plant-pollinator network values. An increase in diversity of insect visitors species significantly increased NMB richness. Furthermore, increased NMB species richness seemed to have a marginal positive effect on conspecific pollen deposition but nor on pollen tube production. Our results suggest that community-level patterns of NMB composition can be determined by the diversity and abundance of the pollinator community, with potential consequences for plant reproductive success.

Introduction: Maternal smoking is a risk factor for childhood overweight and obesity. However, the mechanisms underlying this association are largely unknown. Smoking is associated with changes in the composition of the maternal microbiome and there is now considerable evidence to suggest that the infant microbiome may play an important role in the development of obesity. Therefore we hypothesized that fetal and neonatal exposure to nicotine, the major addictive component of cigarettes, would result in dysbiosis, an alteration in the composition of the microbiome, in postnatal life. Methods: Nulliparous female Wistar rats were randomized to receive daily injections of saline (N=20) or nicotine bitartate (1.0 mg/kg/d; N=20) from 2 weeks prior to mating until weaning. We assessed markers of inflammation, gut permeability, and the composition of the gut microbiota in the offspring. Results: At the phyla level, exposure to nicotine resulted in alterations in the proportion of both Firmicutes and Bacteriodetes at 26 weeks of age. There were significant changes in a number of operational taxonomic units (OTUs) at 3, 12 and 26 weeks of age. Of note, a number of OTUs for Firmicute Clostridia Clostridiales Lachnospiraceae and Firmicute Clostridia Clostridiales Ruminococcus were decreased in the nicotine-exposed offspring which may suggest increased energy extraction in these animals. Although there was evidence of altered gene expression in pathways regulating inflammation and development, these did not result in increased inflammation or aberrant gut development Conclusion: Maternal nicotine-exposure resulted in dysbiosis in the gut of the offspring; an effect that persisted into adulthood. Since dysbiosis has been associated with increased weight gain and adiposity, these data suggest that alterations in the gut microbiome as a result of maternal nicotine-exposure may explain, in part, the increased risk of obesity in children born to mothers who smoke. / Thesis / Master of Health Sciences (MSc)

Background: Studying the infant gut microbiome during the period of solid food introduction may provide valuable insight into gut colonization, microbial evolution, and the ecological role of bacterial metabolic pathways in microbial succession. However, since infant gut microbial communities are made of bacterial genera with high relative abundance, within-genus and within-species diversity, the efficacy of current computational tools in elucidating strain-specific differences is not known. Methods: 34 infant gut metagenomic samples were simulated with the CAMI-Simulator, using 16S rRNA gene profiles from subjects of the Baby & Mi study as a reference. Raw simulated reads were trimmed, assembled, and binned into metagenome-assembled genomes (MAGs) using mg_workflow, a Snakemake-based pipeline of current metagenomic analysis protocols. Results were compared to gold-standard references in order to benchmark the success of current computational methods in retrieving strain-level MAGs from the gut, and in predicting bacterial carbohydrate active enzymes. Real metagenomic samples from the Baby, Food & Mi cohort were processed through the bfm_mg_flow pipeline to study the taxonomic and metabolic changes in the infant gut microbiome during the solid food introduction period. Post-pipeline analyses were conducted in R. Results: Misassemblies were significantly impacted by sample community composition, including Shannon diversity, number of strains in the sample, and relative abundance of the most dominant strain. MAG completeness, contamination, quality, and reference coverage were significantly impacted by choice of assembly software, and choice of single- or co-sample assembly. Different assemblies yielded different MAGs from the same samples. Reference coverage of MAGs recovered from co-assemblies were lower than for those from single assemblies and CAZyme predictions were more accurate from MetaSPAdes than from MEGAHIT assemblies at both the assembly-level and the MAG-level. Based on these results, we propose the MetAGenomic PIpelinE (MAGPIE), with recommendations for ensemble methods for assembly, binning, and gene predictions. Using these methods, we identified changes in microbial community composition before and after solid food introduction in real Baby & Mi infant gut samples. These changes included an increase in bacteria that can digest a wide variety of carbohydrates, such as Bacteroides, and a decrease in Bifidobacterium. Conclusions: In this study, we characterized the current state of tools for genome-resolved metagenomics, and contributed a framework to tailor metagenomic data analysis for the unique composition of the infant gut microbiome. We further used this framework to study bacterial metabolism in the infant gut microbiome before and after the introduction of solid foods. / Thesis / Master of Science (MSc) / Solid food introduction to the infant diet brings new glycans to the gut environment, driving the selection of bacteria that are able to digest these compounds. Studying the gut microbiome during this timepoint is essential to deciphering how and when beneficial bacteria colonize, how they evolve, and how the infant gut matures to an adult-like state. A widely used method to characterize microbial identity and metabolic function in the gut is metagenomic sequencing. However, dominant bacterial genera in the infant gut often have multiple closely related species and strains, making it difficult to decipher the essential metabolic differences between them. In this study, we simulated an infant gut metagenomic dataset to understand how the structure of the infant gut impacts commonly used metagenomic tools, and to quantify the quality of genomes and metabolic predictions at the end of common metagenomic analyses. We found that gut microbial community composition and metagenomic assembler choice both impact the quality of final genomes retrieved from the data, and the accuracy of metabolic gene predictions. Based on these results, we make several recommendations to use ensemble methods to improve metagenomic data analysis, and additionally propose a metagenomic pipeline to analyze infant gut data over the period of solid food introduction.

Most research evaluating the effects of the reproductive tract microbiota on reproductive performance has been done in humans, thus far. In bovids, reproductive microbiota research is not as advanced, with preliminary conclusions, not supported by contamination checks or repeatability. Our studies concluded that endogenous reproductive hormones, days of gestation, and pregnancy status does not change the overall vaginal microbiota composition. Although, the overall composition did not change there were species level differences. These differences could have implications in reproductive performance and fertility in heifers. Heifers that undergo nutrient restriction have similar vaginal microbiota to adequately fed heifers with no species differences. The most impactful finding is that exogenous supplementation of melatonin was associated with changes in the vaginal microbiota in Brangus heifers during late gestation. The implications of this finding are not yet clear, but to date, this is the first hormone, in bovids, determined to change the composition of the vaginal microbiota.

The human gut microbiome is the collection of all organisms and their genetic content that inhabit the gastrointestinal tract. An overwhelming number of studies have associated the gut microbiota with health and disease, but with little consensus on which specific bacterial groups are important for causing or maintaining either state. A majority of microbiome studies only identify associations between the gut microbiome and health status, and determining causation requires the isolation and growth of bacterial isolates for further experiments. The goal of this thesis is to demonstrate that the combination of culture-based and culture-independent methods describes greater complexity and diversity in the human gut microbiota than observed by either approach alone. In the first study, a method of culture-enriched molecular profiling could capture the majority of bacterial groups found in fecal samples. Additionally, when compared to culture-independent 16S rRNA gene sequencing, culture detected more bacterial taxa. This method was applied to the targeted culture of the commensal Lachnospiraceae family. The second study explored the diversity in the isolated Lachnospiraceae strains, and compared the genetic diversity of the strains to reference genomes, revealing functional and genetic heterogeneity within the bacterial family. The third study characterized the intra-species phenotypic and genetic diversity in Escherichia coli. E. coli diversity was extensive between individuals, but also within-individuals, in both the phenotypes and genetic profiles. Lastly, a method of culture-enriched metagenomics was applied to a murine IBS microbiota transfer model to identify bacterial members of the microbiota and their functional pathways that may be responsible for the development of gastrointestinal and behavioural IBS phenotypes, although no bacterial groups could be conclusively associated with symptoms. Together, the work described demonstrates that culture and culture-independent methods are complementary, and provides more resolution into the structure and diversity of the human gut microbiome than either approach in isolation. / Thesis / Doctor of Philosophy (PhD) / Bacteria that inhabit the human intestine are important for health, and are involved in several diseases; therefore, it is critical to determine the roles of specific bacteria. I describe a method that results in the growth and recovery of most bacteria in stool, which allows them to be studied in detail. The differences, both in behaviour and in DNA sequences, found within two different bacterial groups were characterized, and extensive variability was observed between closely related bacteria. I studied which bacteria and their functions might be important in Irritable Bowel Syndrome (IBS) by using our method for growing stool bacteria combined with sequencing of all DNA in the stool, but could not find strong support for specific bacteria causing IBS symptoms. This work shows how the ability to grow and isolate bacteria, combined with studying their DNA, allows for better understanding of their functions in the human intestine.

Multi-drug resistant Pseudomonas aeruginosa has become an increasing threat. A threat compounded by the fact that the pipeline of antimicrobial discovery continues to lose effectiveness. It is urgent that we identify new strategies to eliminate this and other multi-drug resistant pathogens. Meanwhile, we know that the resident bacteria of the respiratory tract interact with host cells to eliminate incoming threats. The goal of this project is to understand the interactions at play in the microbiota of the respiratory tract and identify the specific commensal bacteria that inhibit P. aeruginosa in the presence of host cells. Using a nasopharyngeal cell culture screen, we were able to identify several human commensals capable of inhibiting the growth of antibiotic-resistant P. aeruginosa in a host-dependent manner. It was also established that this phenotype can be reproduced in more complex systems, including a murine lung slice model, and that the elimination of the pathogen is dependent on the presence of living bacteria, mediated by a secreted factor. We believe that the presence of these commensals has an immunomodulatory effect on the host. By characterizing the cytokines and chemokines produced by the host cells in response to the presence of these commensals, we have shown that these bacteria modify the immune response of the host to the pathogen. Finally, having failed to develop a mouse model of infection, we have determined that the observed phenotype is not a direct action of the host cells, nor the result of hydrogen peroxide secretion. We have further characterized the bacterial strains using genome assembly. This study has confirmed that commensal bacteria create a unique immune environment that contributes to the clearance of pathogens. Identifying bacteria and bacterial products capable of stimulating host defenses and modulating inflammation could provide a new therapeutic approach to reducing infection susceptibility in high-risk patients. / Thesis / Master of Science (MSc) / Antibiotics have for decades facilitated the cure of infections caused by pathogens of all kinds. Unfortunately, repeated exposure to antibiotics has rendered some pathogens highly resistant to traditional treatment. One notorious multi-drug resistant pathogen is Pseudomonas aeruginosa, now responsible for 10-15% of hospital-acquired respiratory infections. In this project we investigate the potential that lies in using commensal bacteria that exist in the microbiota of our respiratory tract to fight against pathogens that no longer respond to antibiotics. Stringent experiments with various models have revealed bacteria capable of killing Pseudomonas aeruginosa and helped us understand the mechanism of inhibition as well as the interactions of the bacteria with the immune system. Our results show that some bacteria of the microbiota are effective against Pseudomonas aeruginosa and that this line of study holds promise for the development of a new arsenal against a growing number of threatening pathogens.

Pregnancy loss and calf death can cost dairy producers more than $230 million annually. While methods involving nutrition, climate, and health management to mitigate pregnancy loss and calf death have been developed, one potential influence that has not been well examined is the reproductive microbiome. I hypothesized that the microbiome of the reproductive tract would influence heifer fertility and calf gut microbial development. The objectives of this dissertation were: 1) to examine differences in phenotypes related to reproductive physiology in virgin Holstein heifers based on outcome of first insemination, 2) to characterize the uterine microbiome of virgin Holstein heifers before insemination and examine associations between uterine microbial composition and fertility related phenotypes, insemination outcome, and season of breeding, and 3) to characterize the various maternal and calf fecal microbiomes and predicted metagenomes during peri-partum and post-partum periods and examine the influence of the maternal microbiome on calf gut development during the pre-weaning phase. In the first experiment, virgin Holstein heifers (n = 52) were enrolled over 12 periods, on period per month. On -3 d before insemination, heifers were weighed and the uterus was flushed. Flush pH and glucose concentration were measured. Blood was collected from coccygeal vessels on d -3, 15, 18, 21, 24, 27, and 30 relative to insemination and serum progesterone concentration was measured. Ultrasound measurements of dominant follicle diameter and corpus luteum volume. Insemination outcome was determined on d 30 using ultrasound and pregnancy was checked on d 42, 56, 70, and 84. Heifers were clustered based on outcome of insemination at d 30 (not pregnant, NP30, n = 24; pregnant, PS30, n = 28), d 84 (not pregnant, NP84, n = 24; pregnant but lost before d 84, PL84, n = 2; successfully pregnant through d 84, PS84, n = 26). Differences in phenotypes were assessed based on insemination outcome at d 30 and d 84. Weight was greater in NP30 heifer than PS30 heifers. Progesterone was greater in PS30 and PS84 heifers than NP30 or NP84 heifers on d -3 and 18 to 30 and CL volume was greater in PS30 and PS84 heifers than NP30 and NP84 heifers on d 21 and 30. To summarize, traits related to pregnancy maintenance were different in virgin Holstein heifers based on first insemination outcomes and might be able to be used to predict heifer reproductive performance. Uterine flushes were examined in a subset of heifers (n = 28) based on insemination outcome and period. This subset was also clustered based on season (spring, n = 3; summer, n = 12; fall, n = 8; winter, n = 5). From this subset of heifers, DNA was extracted from uterine flush and 16S amplicons of the V4 region underwent 250 paired-end sequencing via Illumina NovaSeq 6000. Filtered reads were clustered into operational taxonomic units using a 97% similarity and assigned taxonomy using the SSURNA Silva reference version 132. Alpha and beta diversity were measured and differences in alpha and beta diversity measurements were analyzed based on insemination outcome at d 30 or d 84 and season of breeding. Differential abundance analyses were performed at the phylum and genus taxonomic ranks based on insemination outcome at d 30 or d 84 and season of breeding. Bacterial richness was reduced in PL84 heifers than NP84 and PS84 heifers and reduced in heifers bred in spring than those bred in other seasons. Bacterial community structure was different based on insemination outcome at d 30 and d 84 using unweighted Unifrac distances and was different based on season of breeding using weighted Unifrac distances. We observed an increase of Bacteroidetes in PS30 and PS84 heifers compared to NP30 and NP84 heifers. Ureaplasma and Ruminococcus had an increased abundance in PS30 and PS84 heifers than NP30 and NP84 heifers, while Afipia and Gardnerella had an increased abundance in NP30 and NP84 heifers than PS30 and PS84 heifers. Prevotella and Ruminococcus had a reduced abundance in summer bred heifers than winter bred heifers. Proteobacteria had a moderate negative correlation with -3 d progesterone (rp = -0.42) and Actinobacteria had a moderate negative correlation with fetal growth rate (rp = -0.66). Uterine microbiome of virgin Holstein heifers differed based on insemination outcomes and season of breeding and might be a new phenotype to indicate heifer fertility. In the second experiment, multiparous Holstein cows (n = 12) were placed in individual box stalls 14 d before expected calving. Sterile swabs were used to collect samples from the posterior vagina of the dam approximately 24 h before calving, dam feces, dam oral cavity, and colostrum within 1 h after calving, and cotyledonary placenta within 6 h after calving. Calves (n = 12; bulls = 8, heifers = 4) were isolated immediately after parturition to prevent environmental contamination. Colostrum was fed to calves using a clean bottle that was assigned to the calf for the duration of the study. Calves were individually housed for 60 d until weaning. Sterile swabs were used to collect calf fecal samples at birth, 24 h, 7 d, 42 d, and 60 d of age. A subset of calf-dam pairs (n = 6; bulls = 3, heifers = 3) were selected and DNA was extracted from all samples. Amplicons covering V4-V5 16S rDNA regions were generated using extracted DNA and sequenced using 300 bp paired end sequencing via Illumina MiSeq. Sequences were aligned into operational taxonomic units using the 97% Greengenes reference database. Spearman correlations were performed between maternal and calf fecal microbiomes. Negative binomial regression models were created for genera in calf fecal samples at each time point using genera in maternal microbiomes. Metagenomes were predicted, collapsed into gene pathways and differences in predicted metagenomes were analyzed within STAMP (Statistical Analysis of Metagenomic Profiles). We determined that Bacteroidetes dominated the calf fecal microbiome at all time points (relative abundance ≥ 42.55%) except for 24 h post-calving, where Proteobacteria were the dominant phylum (relative abundance = 85.10%). Colostrum and placenta had low diversity within samples and clustered independently from fecal samples. Each maternal microbiome was a significant predictor for calf fecal microbiome during at least 2 time points. Genes for infectious disease and neurodegenerative disease were greater in colostrum and 24 h calf fecal samples compared to other samples. Results indicated that no one maternal microbiome was a major influence on calf fecal microbiome inoculation and development. Instead, calf fecal microbial development stems from various maternal microbial sources. Overall, the reproductive microbiome was predictive of heifer pregnancy outcomes and calf fecal microbial development. The virgin heifer uterine microbiome could be used to predict fertility and adaptation to heat stress, but further research including a larger group of pregnancy loss is needed. Maternal microbiomes from the reproductive tract, colostrum, oral cavity, and feces could all be used to predict calf microbial development, but more research including other maternal microbiomes and environmental microbial contributions is needed. However, the results from this dissertation indicate reproductive microbiome composition is a trait that might be predictive of dairy cattle performance. / Doctor of Philosophy / The ability of a cow to become pregnant and a calf to thrive after birth are crucial to successful dairy farm operations. Recent evidence in humans has shown bacteria in the reproductive tract can influence maternal fertility and the bacterial community of newborns, an indicator of early health. This same relationship might exist in dairy cattle. I propose that specific traits related to fertility and the bacterial community in the reproductive tract of dairy cattle influences their ability to become pregnant and influences the bacterial community developing in calves after their birth. In my first experiment, I collected samples of uterine fluid from cattle that had never been pregnant before the first time they would be bred. I also collected blood samples before and after breeding to measure hormone levels as well as measurements of portions of reproductive tract using an ultrasound. Using a specific portion of DNA that is similar across all bacteria, I identified the bacterial community in the collected uterine fluid. Cattle were grouped based on breeding outcome (not pregnant, pregnant but lost, or kept pregnancy) and season of breeding. Differences in various traits and bacterial communities were examined based on breeding outcome and season. I found that traits like hormone levels in the blood and size of structures on the reproductive tract, and uterine bacterial community all differed based on breeding outcome. We also found that uterine bacterial community also differed based on season of breeding. These results could be used to predict if a cow will become pregnant before they are ever bred, but more research is needed. In our second experiment, we collected samples from the reproductive tract, milk, mouth, and feces of cows immediately after they gave birth. We then collected samples from their calves right at birth as well as at various time points during their early life. Using the same section of DNA used during the first experiment, we identified the bacterial community composition from the various maternal and calf samples. We then identified correlations between maternal and calf bacteria and used a mathematical model to see if the maternal bacteria could predict bacteria in the calf. We found that the various maternal bacteria could predict calf bacteria throughout the calves early life. While an experiment using a larger group of cows and calves is needed, our results indicate that the maternal bacteria could be used to predict calf bacteria and may help determine which calves are more likely to become sick than others. Overall, we found that the bacteria in the reproductive tract could be used to predict ability to become pregnant and calf bacterial development. The incorporation of this bacterial community as a trait on farms could help reduce pregnancy loss and calf illness, but further research examining how the bacteria interact with the animal is needed.

The year 2020 marked one of the hottest years on record to date, with the average global temperature reaching 1.2 °C above pre-Industrial era (1880) temperatures. Rising temperatures are largely attributed to increasing CO2 levels from the widespread burning of fossil fuels. Terrestrial ecosystems make up the largest global carbon reservoir. In the soil, microorganisms play major roles in carbon and nutrient cycling, decomposition, and mediation of plant health, among several others. Involvement in such important processes makes soil microbial communities incredibly insightful for understanding earth’s changing climate. The Harvard Forests in Petersham, MA implement belowground heating cables to warm experimental soils 5°C above the ambient soil temperature. With this dramatic temperature difference, researchers intended to simulate a worst-case scenario for earth’s climate. However, upward trends in global warming make this projection not as far out of reach as originally thought. In 2017, the heating cables in experimental soil plots were turned off after approximately 15 consecutive years of warming and the soil was allowed to re-equilibrate to the ambient temperature. Experimental soils took around 2 months to reach the same temperature as the control soils. Significant changes in soil respiration levels and moisture were observed, raising the question as to whether soil microbial gene expression levels changed as well. Soil samples were collected for bulk RNA extraction from both heated and control soil plots on the day the heating cables were turned off (Day 0) and sequenced on the Illumina NextSeq platform at the Department of Energy’s Joint Genome Institute. Here I present a metatranscriptomic analysis of the Harvard Forest soil microbiome on Day 0 to uncover the soil microbial community’s transcriptional response to long-term warming. Major phyla that were less transcriptionally active in response to warming include Basidiomycota, Pseudomonadota, Bacteroidetes, and Acidobacteria, which have essential roles in decomposition, nutrient cycling, mediating plant health, and more. Phyla that were more transcriptionally active in response to warming include Actinobacteria, Ascomycota, and Chloroflexota, which participate in biogeochemical cycling, polymer breakdown, antimicrobial activity, and more. These changes in activity reflect the ways in which the soil microbiome responds to chronic warming.