With a growing human population, food insecurity is becoming a worldwide problem. As the search for sustainable sources of protein continues, black soldier fly larvae come to the forefront as a partial solution. Full of proteins and fats, the larvae will consume most organic matter and rapidly develop into a usable form. Supplementing black soldier fly larvae with oleaginous microbes Arthrobacter AK19 and Rhodococcus rhodochrous increases their body size by 25%, potentially accelerates their development by one day, and increases their conversion efficiency. Supplementing with Bifidobacterium breve decreased body size, slowed development, and decreased conversion efficiency, underscoring the importance of selecting supplemental microbes and testing first on a small-scale. Promising results on the small-scale led to an industrial study, where similar results were also seen, resulting in greater optimization of this system.

Microbiomes are an extremely vast and complex network of microorganisms. Bacteriophages are a key factor in the microbial health of an ecological system and impact the evolution of pathogenic bacterial strains. Bacteria and the phages that infect them have an intricate relationship due to the dependency on the bacterial host for phage replication, the ability of the phage to lyse and kill its host, and the horizontal gene transfer between the host and phage. This thesis aimed to understand how bacteria and the bacteriophages that infect them impact an ecological system, with a focus on disease states. By analyzing all bacteriophages targeting a specific host, genomic properties, physical similarities and differences a better understanding of how a group of tailed phages have evolved numerous mechanisms and tools to infect host bacteria was understood. The microbiome study of the Western honey bee Apis mellifera, comparing the microbial communities of colonies infested with the external parasite Varroa destructor against those not infested revealed a need for more directed treatment of Varroa infestations. Through our study we discovered that the honey bee microbiome is much more complex than previously reported, consisting of hundreds of bacterial species. In addition, through comparing infested and healthy colonies, we discovered that infestation of Varroa destructor mites negatively impact the colony microbiome in part by reducing microbes key in digestion and immune health of honey bees. Results in this thesis indicate that two microbes which have not been previously established as part of the key microbes in honey bee guts, Xenorhabdus and Sodalis, may in fact be key to honey bee health as they were both effected negatively by the presence of Varroa mite infestations. These bacteria have been shown to be involved in immune health in other insects, supporting this hypothesis. The final stage of this thesis involved the development of an acaricide bacteriophage therapy designed to target key microbes in the gut of V. destructor. Our therapy was shown to safely treat honey bee colonies infested with this parasite. It can be concluded that while phages are a normal part of the microbial ecosystem of the intestinal tract of organisms, altering that balance by increasing the phage load on the microbiome to target specific beneficial microbes drastically reduces the overall fitness of the organism. Results from this study indicate that multi-target cocktail and single-target phage therapy are an effective low impact biological Varroacide. The discovery of an effective therapy is important and to better understand the results of this thesis, further investigation is required, including a study of the impact of the phage therapy on the mite microbiome, as well as the safety of the therapy to humans.

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In this work we use the fruit fly Drosophila melanogaster as a model to identify bacterial genes that help bacteria to persist in their animal hosts. Early work on this model system established that dietary replenishment drives the composition of the D. melanogaster gut microbiota, and subsequent research has shown that some bacterial strains can colonize the fly for much longer than the flow of bulk diet through the gut. In this work we reveal that bacterial genes influence bacterial persistence by studying the correlation between bacterial genotype and persistence in the D. melanogaster gut microbiota. We performed an initial assay with 7 bacterial strains to establish that different bacterial strains persist differently independent of ingestion in the fly. We then repeated the assay with 41 different strains of bacteria in order to perform a metagenome wide association (MGWA) to find distinct bacterial genes that are significantly correlated with persistence. Based on the MGWA, we tested if 44 mutants from 6 gene categories affect bacterial persistence in the flies. We identified that transposon insertions in four flagellar genes (fliF, flgH, fliI, and flgE), one urea carboxylase gene, one phosphatidyl inositol gene, one bacterial secretion gene, and one antimicrobial peptide (AMP) resistance gene each significantly lowered colonization forming units (CFUs) that resulted from plating the gut content in Drosophila melanogaster. Follow-up experiments with the flagellar gene mutants revealed that each significant flagellar mutant was non-motile compared with the wild type. Taken together, these results reveal that there are bacterial genes that are involved in mechanisms, like bacterial motility, that help bacteria to persist in the fly gut.

The importance of the gut microbiome is being explored in relation to multiple facets of health and performance. The purpose of this study was to explore the impact of frequent sweet potato ingestion on alterations of the gut microbiome which might in turn alter cholesterol levels and exercise performance. Thirteen recreationally trained males had fecal and blood samples collected and exercised to fatigue with and without exogenous carbohydrate ingestion prior to and following 42 days of daily sweet potato ingestion. Bacterial communities were extracted from fecal samples and bacterial DNA were sequence. Blood samples were analyzed for total cholesterol, low-density lipoprotein-C, high-density lipoprotein-C, and triglycerides. Performance was analyzed comparing changes in time-to-volitional fatigue with and without carbohydrate ingestion. Changes were observed in microbial abundance following 42 days of sweet potato ingestion but no differences in cholesterol or cycling fatigue with and without carbohydrates.

Introduction: Short- and long-term health outcomes of children stem from their first 1000 days of development (3 months prior to conception to 2 years postpartum). Research shows a correlation between poor maternal nutrition and adverse birth outcomes. Various factors such as human breast milk (HBM), gut microbiome (GM), and body mass index (BMI) correlate with nutrition. The purpose of this study was to determine if maternal factors such as BMI impact preterm infant microbiome and short chain fatty acid (SCFA) profiles. Methods: Sample Collection: In order to understand the effect of maternal health factors on neonatal GM, deidentified stool samples were collected from the NICU at the MUSC and were utilized for GM and SCFA analysis at ETSU. Microbiome Analysis: GM analysis was performed on stool samples using the Qiagen QIAmp PowerFecal Pro DNA Kit. DNA was sequenced using Amplicon sequence of the 16s rRNA region with a modified Klindworth et al method. GM was analyzed using CLC Genomics Workbench v. 23 where Alpha diversity indexes were calculated with the Abundance Analysis tool and the Beta diversity (inter-sample diversity) was calculated using the weighted Unifrac metric. Short Chain Fatty Acid Analysis: The stool samples were subjected to SCFA extraction and analysis via a modified Schwiertz et al. method. Results: Significance was observed between the groups in microbiome for; C-section (yes, no), gestation (<28, 28-32, 33-36 weeks), week of sample collection (1, 2, 3, 4, >4 weeks), and maternal BMI + antibiotics (no antibiotics + normal, overweight, or obese BMI and antibiotics + normal, overweight, or obese BMI). Significance was detected between the groups in fecal fermentation for; recreational drug use (use, no use), preeclampsia (preeclamptic, not), sepsis evaluation (yes, no), week of sample collection (1, 2, 3, 4, >4 weeks), and Fenton measurements for birth length, birth weight, and occipital frontal circumference (small, average, large for gestational age). Conclusions: These results provide valuable insights into the various maternal and neonatal factors on the GM and SCFA profiles of preterm infants, which can have implications for their overall health and development. It is possible for future adverse health outcomes of premature neonates to be attenuated through HBM ingested and GM.

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