Research in our lab has predicted hundreds of bacterial genes that influence nine different traits in the fruit fly, Drosophila melanogaster. As a practical alternative to creating site-directed mutants for each of the predicted genes, we created an arrayed transposon insertion library using a strain of Acetobacter fabarum DsW_054 isolated from fruit flies. Creation of the Acetobacter fabarum DsW_054 gene knock-out library was done through random transposon insertion, combinatorial mapping and Illumina sequencing. Successful mapping of transposon insertion was achieved for 6418 mutants with hits within 63% of annotated genes within Acetobacter fabarum DsW_054. Insertion sites were verified in 40 mutants through arbitrary PCR and sequencing. To test the utility of the library, genes were selected from MGWAS results on host colonization which show LPS pathway enrichment in the significant gene predicctions. Genes upstream of Lipid-A creation show significant differences in host colonization whereas downstream genes show no effect. In addition, genes were selected from MGWAS results on Drosophila starvation resistance which show Methionine/Cysteine synthesis, Cobalamin synthesis, and Biotin synthesis pathway enrichment. Under our experimental conditions we could not verify influence of these pathways on host starvation resistance. However, they do appear to influence host colonization abundance. This transposon insertion mutant library will be useful for ongoing research in our lab as well as any field studying Acetobacter species, such as other insect microbiome and fermentation research.

Increased exposure to antibiotics has led to the dissemination of genes conferring resistance to antimicrobial metabolites throughout human microbiomes globally via horizontal gene transfer (HGT). This has resulted in the emergence of new resistant strains leading to a rising epidemic of deaths from previously treatable infections. Evidence suggests that before the age of anthropogenic antibiotic use, microbes living within a community produced antibiotic metabolites and, subsequently, maintained such genes for several useful functions and a balance of diversity in nature. The question of the origin of these resistant genes is difficult to answer, but with continued advancements in ancient genomic analysis, researchers have developed methods of acquiring a more accurate representation of the microbiome associated with our human ancestors by extracting fossilized microbial specimens from dental calculus and directly sequencing the metagenomes. This thesis outlines the production of taxonomic and functional profiles of 20 different human and non-human oral microbiome samples using metagenomics tools originally developed for living individuals, altered for use with ancient microbial specimens. Putative antimicrobial resistant (AMR) genes derived from these profiles were reconstructed and conserved functional regions were identified. From the data that is available regarding the human microbiome from a range of time points throughout history dating back to Neanderthal specimens, it is possible to elucidate relationships between these AMR genes and to better understand the evolutionary trajectory of antibiotic resistance.

No description available.

Autism spectrum disorder (ASD) is a neurodevelopmental disorder marked by social deficits and repetitive actions. A communication pathway exists between the brain and gut called the gut-brain axis. It is thought that gut bacteria can secrete signaling molecules, triggering inflammation across the body. These studies attempt to determine if markers are expressed in two mouse models of ASD behaviors, BTBR and a valproic acid model. Immunohistochemistry of ionized calcium binding adaptor molecule 1 from male mouse brain tissue showed no microglial activation in any group. Cytokine analysis did exhibit an increase in interleukin 1 (IL-1) in male adult mice only. Sequencing of bacterial profiles demonstrated differences between groups. Altogether, it appears that microbiome differences do not trigger robust differences in inflammatory pathways in these animals in this study. It is imperative that a reliable animal model of behaviors be identified for novel studies that can impact the development of the disorder.

abstract: Anoxia tolerance is strongly correlated with tolerance to heat, desiccation, hyperosmotic shock, freezing, and other general stressors, suggesting that anoxia tolerance is broadly related to stress tolerance. Age affects the capacity of many animals to survive anoxia, but the basis to this ontogenic variation is poorly understood. We exposed adult Drosophila, 1, 3, 5, 7, 9, and 12 days past eclosion, to six hours of anoxia and assessed survival 24-hours post-treatment. Survival of anoxia declined strongly with age (from 80% survival for one-day-old flies to 10% survival for 12 day-old-flies), a surprising result since adult fly senescence in Drosophila is usually observed much later. In anoxia, adenosine triphosphate (ATP) levels declined rapidly (< 30 min) to near-zero levels in both 1 and 12-day old adults; thus the higher anoxia-tolerance of young adults is not due to a better capacity to keep ATP elevated. Relatively few physiological parameters are reported to change over this age range in D. melanogaster, but gut bacterial content increases strongly. As a partial test for a causal link between bacterial load and anoxia tolerance, we replaced food daily, every third day, or every sixth day, and assayed survival of six hours of anoxia and bacterial load at 12 days of age. Anoxia tolerance for 12-day old flies was improved by more food changes and was strongly and negatively affected by bacterial load. These data suggest that increasing bacterial load may play an important role in the age-related decline of anoxia tolerance in Drosophila. / Dissertation/Thesis / Masters Thesis Biology 2020

Aquaculture is currently the fastest growing animal production sector. Because the aquaculture sector is growing at rapid rates, certain materials for feed, specifically marine protein sources, are becoming increasingly expensive and unsustainable. To counteract the reliance on fishmeal (FM) and other marine protein sources in the industry plant protein (PP), specifically soybean meal (SBM), has been investigated to replace FM as a protein source. Unfortunately, SBM when given in high quantities (greater than 30%) has been shown to negatively affect fish performance including retarded growth, intestinal inflammation, reduction of spawn quality, as well as dysbiosis in the gut microbiome, most likely due to presence of antinutritional factors such as saponins and tannins in SBM. The goal of this thesis was to investigate the effect of nutritional programming (NP) with SBM-based diet on gut microbiota in broodstock and progeny fish. Three feedings trials were conducted to test the efficacy of 3 approaches towards improving the use of PP in fish.The first trial (Chapter 2), tested the effect of NP on larval zebrafish (Danio rerio). NP is the theory of introducing an early nutritional stimulus to an animal that will ‘program’ the animal to better utilize the stimuli later in its adult life. The zebrafish were programmed in their larval stages, and the trial lasted for 65 days. There was a significant effect on growth performance for the programmed group (NP-PP) in terms of weight gains, as the NP-PP group grew better compared to the non-programmed group (NP-FM) and negative control (-control). There was no significant effect on the gut microbiome in terms of alpha or beta diversity, however, there were significant changes in the relative abundance (RA) of the gut microbiome throughout time in the NP-PP and the NP-FM groups. The findings of the study support that early NP of zebrafish with SBM improves growth performance on PP diet, but the gut microbiome does not seem to be a mechanism for NP.The second feeding trial (Chapter 3) focused on NP induced in the zebrafish broodstock with dietary SBM. For two weeks, the broodstock fish were fed with either a SBM diet or a FM diet so that gametogenesis occurred with either a FM or PP diet. This phase was called the ‘broodstock programming’ stage. The broodstock were then spawned, and the larval fish were separated into four different treatments: 1) SBM broodstock progeny, fed SBM for the entire trial (PPBS-PP) 2) SBM broodstock progeny fed FM the entire trial (PPBS-FM), 3) FM broodstock progeny fed FM the entire trial (+ control, FMBS-FM), and 4) FM broodstock progeny fed SBM the entire trial (- control, FMBS-PP). The PPBS-PP group achieved similar weight gains compared to all other treatments in terms of grams, but was numerically greater than the FMBS-PP treatment. There were no differences detected in gut microbiome alpha or beta diversity in any of the groups, however, there was significant change observed of certain bacterial phyla between the ‘programmed broodstock’, larval fish, and fish at the end of the trial, 48 days post hatch. Overall, this trial suggests that parental programming does not improve PP utilization in the progeny of zebrafish. It also appears that the gut microbiome is not a mechanism of parental programming. The third feeding trial (Chapter 4), was conducted on largemouth bass (Micropterus salmoides). This chapter had a similar experimental design as the first trial (Chapter 2), and larval largemouth bass were programmed with dietary SBM. This trial had an additional group added to it, which included a dietary saponin-programmed group. The study found that the NP with SBM diet or dietary saponin did not improve PP utilization and growth performance of largemouth bass in its pre-adult age. The study also found that the NP with SBM diet or dietary saponin did not have any effect on the largemouth bass gut microbiome, and there does not seem to be any gut microbiome modification associated with the NP in this fish species. Overall, NP can be used to improve dietary PP utilization but optimal timing and PP delivery method must be well assessed to ensure successful PP exposure and adaptation in different species. Nevertheless, the gut microbiome does not seem to be affected by NP and therefore is not considered the mechanism behind NP. Finally, studies on both zebrafish and largemouth bass presented major shifts in the gut microbiome as the fish aged. In addition, the core microbiomes of both species appeared to become more pronounced as the fish become adults. There seem to be an evolutionary tie between host and its gut microbiome. More studies, however, should further investigate this and the genetic effects on gut microbiota development and its heritability.

<p>Carbohydrases have been used to improve fiber utilization in monogastric animals. However, their effects on animal performance and nutrient digestibility have been inconsistent in pigs. The efficacy of carbohydrases has been suggested to depend on enzyme activity and fiber composition, but recent evidence suggests other factors like environmental conditions may play significant role. The effect of heat stress (HS) on the efficacy of a multienzyme carbohydrase blend in growing pigs was investigated. It was hypothesized that HS alters the efficacy of carbohydrases. Ninety-six growing pigs (gilts:barrows; 1:1) (initial BW of 20.15±0.18 kg) were randomly assigned to 6 treatments, with 8 replicates of 2 pigs replicate pen in a 3×2 factorial arrangement: three levels of carbohydrase (0, 1X or 2X) and two room temperature conditions (20^oC constant or cyclical 28^oC night time and 35^oC daytime). The 1X is the recommended commercial dose of the enzyme (50g/tonne) and provides 1250 visco-units endo-β-1,4-xylanase, 4600 units α-L-arabinofuranosidase and 860 visco-units endo-1,3(4)-β-glucanase per kg of feed. The 2X dose was 100g/tonne. Pigs were fed ad libitum for 28 days and 1 pig per pen was sacrificed at d28. Heat stress increased the respiratory rate and skin temperature (P<0.001). There was no enzyme × temperature effect on response criteria. Enzyme treatment quadratically increased BW d28 (P=0.025), ADG (P=0.022) and average daily feed intake (ADFI) (P=0.032) with 1X being the highest. Heat stress reduced the BW at d14 (P=0.002) and d28 (P=0.006), average daily gain (ADG) (P=0.005) and ADFI (P<0.001). However, there was a trend of increased gain: feed ratio (G/F) (P=0.093) in the HS pigs compared to the thermoneutral (TN). Heat stress increased apparent jejunal digestibility (AJD) of energy (P=0.039) and apparent ileal digestibility (AID) of calcium (P=0.007). ADFI was positively correlated to ADG (r=0.57; P<0.001) but negatively correlated to G/F (r=-0.42; P=0.003). Enzyme supplementation increased in vitro viscosity at 3, 4 and 5 hours (P value). Heat stress reduced serum glucose concentration on d1 (P=0.0002) but increased serum non esterified fatty acid (NEFA) concentration (P=0.002). A similar trend to NEFA was observed in triacylglycride (TAG). In the jejunum, carbohydrases had trend for increased villi height (P=0.07) while HS reduced villi height (P=0.02). Heat stress increased the jejunal mRNA abundance of IL1β in the jejunum (P<0.001). There was a trend for a reduction in ileal MUC2 (P=0.092), occludin (P=0.091) due to HS, with the trend increasing in PEPT1 (P=0.064). There was no effect of HS on alpha diversity of fecal microbiome, but sampling day affected beta diversity. There was an increase in the abundance of pathogenic bacteria (like Clostridium) in the HS group. Heat stress did not alter the efficacy of carbohydrase but both carbohydrase and HS modulate pig performance independently.</p>

Depression and anxiety disorders affect upwards of one in six individuals at some point in their life making them the most prevalent mental illnesses today. Recent evidence has suggested a possible correlation between the human gut microbiota and the development of depressive and anxiety-like symptoms through a signaling pathway termed the microbiota-gut-brain axis. In both animals and individuals suffering from depression and anxiety-like symptoms, alterations in their gut microbial composition seem to compromise the function of this pathway. In addition to this microbiota-gut-brain axis, other microbiota-derived molecules have been linked to symptoms of depression and anxiety. Given this emerging role of the gut microbiome and gut–brain axis, it is crucial to understand the factors shaping our gut microbiome in order to determine potential therapeutic strategies to treat depression and anxiety. Following a concise review of the human microbiome, depression/anxiety, and the gut-brain axis, I will examine the gut microbiota role as a regulator of depression and anxiety. In addition, other biological markers associated with both the gut microbiome and these disorders will be reviewed. Lastly, I will evaluate the gut microbiome as a prospective therapeutic target for mental illnesses such as depression and anxiety.

The human vagina is unlike that of any closely related phylogenetic species due to the abundance of a lactic acid producing bacteria, known as Lactobacillus. This microbial species is known for its direct and indirect contributions to vaginal pathogenic defense, some of which include the elicitation of host immunomodulators, release of bacteriocins and biosurfactants, and lowering of vaginal pH. While Lactobacillus is often considered the hallmark of a healthy human vagina, a significant number of women worldwide lack its abundance in their vaginal microbial communities. The lack of Lactobacillus- dominance does not necessarily equate to a disease-state but could potentially explain an increased risk of viral transmission and recurrent vaginal dysbiosis in these women. This thesis aims to investigate the numerous antimicrobial properties associated with the Lactobacillus species to better understand its significance for female reproductive and vaginal health.

Objectives The gut microbiota and its metabolites – namely short chain volatile fatty acids (SCVFAs) – interact with the digestive, immune, and nervous systems. States of microbiome dysbiosis are highly associated with obesity and GI symptoms, and profiles of SCVFAs, which serve functions as fuel sources and signaling molecules, mimic this dysbiotic state. This study aimed to further our understanding of associations between bacterial diversity and GI symptoms, BMI, immune markers, and SCVFAs and to identify bacteria differentially abundant with changes to the previously mentioned variables. Methods Data (measures of GI distress, BMI, immunoglobulins, fecal proximate analysis, SCVFAs, and 16s RNA sequences) was extracted from a study containing non-celiac gluten-sensitive and control participants. QIIME2 was used to process 16s RNA data, analyze quantitative, qualitative, phylogenetic quantitative, and phylogenetic qualitative measures of alpha and beta diversity and to perform an analysis of composition of microbes (ANCOM) for differential abundances data. Results Many significant differences were seen, namely in multiple measures of alpha diversity for IgG4 (P < 0.018), propionate (P < 0.014), caproate (P < 0.003), heartburn (P < 0.004), urgent need to defecate (P < 0.027), and feelings of incomplete evacuation (P < 0.024). Statistical significance was seen in multiple measures of beta diversity between normal and overweight (P < 0.01), normal and obese (P < 0.005), and overweight and obese BMI (P < 0.016), IgG4 (P < 0.033), propionate (P < 0.001), increased gas (P < 0.024), and urgent need for defecation (P < 0.026). The ANCOM identified multiple species of bacteria differentially abundant with changes to variables. Conclusions Findings suggest differences in both alpha and beta diversity with various GI symptoms, SCVFAs, and BMI. This research supports plans to apply analysis to larger sample sizes to train machine learning classifiers to identify important features of microbial profiles associated with certain SCVFA and markers of health. Funding Sources ETSU (CCRHS, Deans's Research Enhancement Award and CPH, Health Sciences Funding, Honors College Summer Research Fellowship) and Shield Nutraceuticals, LLC.