Within hemipterans, the acquisition of bacterial symbionts has helped insects utilize the nutrient-limited resource of plant sap. Many of these bacterial symbionts have undergone a dramatic reduction in genome size, often losing key regulatory genes. It is therefore unclear how and if these symbionts regulate their gene expression. My dissertation explores the potential role that symbiont expressed small RNAs (sRNAs) have in gene expression. First, I use RNA-seq to characterize the sRNA expression profile of Buchnera, during two different life-stages (aphid ovarioles and maternal bacteriocytes) in which Buchnera has differential protein expression. The results from this experiement show that Buchnera sRNAs are differentially expressed between life-stages. My dissertation also provides in vitro evidence of the functionally of the Buchnera antisense sRNA carB. These results suggest that when Buchnera is in an extracellular state, free of the bacteriocytes, it can respond to changes in host nutritional demand. I then characterized Buchnera’s sRNA expression when its aphid host fed on two different host-plants which have been shown to have different nutritional and plant defense profiles. The results from this experiment show that Buchnera sRNA expression varies with aphid host-plant diet. These results suggest that Buchnera sRNAs can potentially impact the symbiosis in an adaptive nutritional manner, or stress response manner when aphids feed on a host-plant that is lower in nutrients. I also determined that sRNAs are expressed and conserved in one of the most reduced obligate insect symbionts, Candidatus Carsonella ruddii. Currently, many of the functional genomic tools that are optimized to work in a handful of model systems. As result, when working with non-model, unculturable systems, there is an additional challenge of optimizing and modifying current functional genomic tools. The last part of my dissertation tests the efficacy of novel RNAi delivery systems within three aphid species. This RNAi delivery system has resulted in successful gene knockdown in the soybean aphid. Overall, the findings of my dissertation strongly support the hypothesis that small bacterial genomes may utilize sRNAs to help regulate their own gene expression to help compensate for the loss of canonical regulatory proteins.

In this dissertation, the integrated metabolism of three aphid species (Acyrthosiphon pisum, Aphis glycines, and Myzus persicae) and their specialized endosymbionts was explored. In the first chapter, tissue- and host plant-specific profiles of gene expression and CpG methylation of A. pisum were analyzed. Through RNA-Seq and whole genome bisulfite sequencing, I identified key metabolic genes that are differentially expressed and methylated between bacteriocytes and body cells. Moreover, I demonstrated for the first time that key aphid genes involved in the regulation of aphid-Buchnera symbiosis are differentially expressed and methylated depending on the aphid’s host plant diet, suggesting that DNA methylation may be a key regulatory factor that induces phenotypic variation depending on the host plant diet. In the second aphid system, I empirically and computationally confirmed the functional CpG methylation system in Aphis glycines. Also, I showed that lineage-specific genes in A. glycines have significantly lower CpG methylation levels compared to evolutionarily conserved genes. Moreover, five aphid-specific genes were identified to play key roles in insect-plant interactions at the epigenetic level. In the last aphid system, I identified differentially expressed genes in bacteriocytes of Myzus persicae compared to its body cells. I demonstrated overall up-regulation of the genes that are involved in amino acid biosynthesis as well as key genes in aphid-Buchnera integrated metabolism. I then compared gene expression patterns of M. persicae bacteriocytes to those of A. pisum bacteriocytes. I found that the two closely related species have very similar gene expression profiles in their bacteriocytes while there are lineage-specific expression signatures in some metabolic genes.

The intestinal interface allows the host to incorporate and respond to signals from the environment. One way the intestinal contents communicate with the host is through the aryl hydrocarbon receptor (AHR). Highly expressed in the intestinal epithelium and the underlying lamina propria immune cells, the AHR is a cytosolic transcription factor that alters cellular function in a location and cell-dependent manner. Given the evidence that the intestinal environment is dysregulated in type 1 diabetes (T1D), an autoimmunity disorder in which the pancreatic beta cells are attacked, this dissertation examines how modulating AHR signaling in intestinal immune cells may alter diabetes development using the nonobese diabetic (NOD) mouse model of T1D. Chapter 1 is a literature review of AHR signaling in macrophages. The chapter covers what is currently understood about AHR signaling in different contexts, including macrophage differentiation, polarization, function, and immune-mediated diseases. Chapter 2 examines how AHR modulates the gut microbiome during type 1 diabetes development and the role of intestinal CD4+ T cells. Flow cytometric analysis of intestinal CD4+ T cells and 16s microbial data were analyzed from of AHR WT and KO NOD mice at three different time points. Additionally, we utilized specific pathogen-free (SPF) and germ-free (GF) mice to understand the impact of the microbiome on the intestinal CD4+ T cells and diabetes incidence. To investigate how the intestinal T cells interact with the microbiome, we utilized Transkingdom network analysis.

Chapter 3 focuses on the mechanisms occurring upstream of CD4+ T cells in AHR KO NOD mice. Specifically, this chapter identifies intestinal macrophages as a critical target of AhR signaling in the small intestine, utilizing flow cytometry and single-cell RNA sequencing. We then performed experiments using an AHR ligand-enriched diet to demonstrate that macrophages are regulated in an AHR-dependent manner. We provide bioinformatic and functional evidence that suggests these cells interact with intestinal CD4+ T cells in AHR KO NOD mice. Lastly, we used bulk RNA sequencing of intestinal and pancreatic CD45+ cells to perform causal inference analysis to predict how AHR signaling impacts inter-organ communication. Chapter 4 examines the role of AHR signaling in vitro using bone marrow-derived macrophages. Additionally, immunotoxicity of per-and-poly-fluoroalkyl substances (PFAS), ubiquitous environmental pollutants which have been associated with autoimmunity development, is assessed in vitro. By exposing macrophages derived from NOD and B6 mice to PFAS, we model the impact of PFAS exposure in individuals genetically susceptible to T1D. Collectively, the chapters demonstrate that intestinal immune cells are altered by AHR signaling and modulation through AHR signaling may represent a novel mechanism in which the environment influences diabetes development. Additionally, the PFAS study demonstrates that cytokine production is affected by exposure and therefore may alter the host immune response.

Microbial symbionts in animals affect various processes and can be mutualistic, commensal, or parasitic. Symbionts can be obligate or facultative, depending on their requirement for host survival. Insects, particularly herbivores, often form intimate associations with symbiotic microbes involved in nutrient synthesis and plant material digestion. Psyllids, a group of sap-feeding insects, are known to associate with obligate symbiont "Candidatus Carsonella ruddii," which synthesizes essential amino acids for the psyllid's nitrogen-poor diet. Psyllids also harbor facultative symbionts from diverse lineages, horizontally transmitted and prevalent in many species. In psyllids, some facultative symbionts act as both plant pathogens and endosymbionts, such as Candidatus Liberibacter species, which circulate throughout the psyllid's body and may benefit the psyllid by suppressing plant defenses. Despite growing attention to microbial communities in insects, the dynamics of psyllid microbial communities and the interactions between obligate and facultative symbionts remain unclear. My dissertation explores a variety of aspects of the psyllid microbiota, particularly the Liberibacter-Carsonella-host interface. In chapter 2, using 16S rRNA amplicon sequencing and metagenomic sequencing, I revealed potential long-term psyllid symbiont associations in the family Triozidae and Psyllidae. I have also discovered a novel Ca. Liberibacter spp., Ca. L. capsica, which is potential pathogen of solanaceous crops. These findings suggest microbiome profiling of psyllids has great potential for revealing potential risk of new pathogen development and regulatory concerns. In Chapter 3 and 4, taking advantage of the high-quality chromosomal genome assembly and transcriptomes from distantly related species, Diaphorina citri and Bactericera cockerelli, I investigated conserved and lineage-specific gene expression patterns that are related to nutritional symbioses, Carsonella. In the last part of my dissertation, I demonstrated how Ca. L. psyllaurous, and host plant use can influence the integrated metabolism of Carsonella and B. cockerelli. Ultimately, this study has yielded valuable insights that will serve as a foundation for future research on Ca. L. psyllaurous-Carsonella-psyllid interactions.

A mounting body of health care research reports that American retirees are likely to face large and increasing expenditures for out-of-pocket health care costs and that they often struggle to finance these expenditures.  It is, however, unclear whether the general population understands what their likely out-of-pocket health care expenditures might be in retirement.  It is even less clear whether the population appreciates how much these costs can vary from person to person and over time.  It is critically important to know whether Americans understand these costs and to what degree misunderstanding might hamper their financial planning  for retirement, especially in light of recent discussion of Medicare reform that could affect these costs.

This article explores this previously unexamined question of how much Americans expect to pay for their out-of-pocket health care spending in etirement.  To answer this question,we surveyed 2000 near retirees and retirees to gauge their expectations with regard to their own likely expenditures.  We then compared their responses to experts’ estimates. Our findings suggest that, surprisingly, many respondents have a reasonable sense of the magnitude of likely out-of-pocket expenditures, at least for the median, or typical, retiree.  However, we also found people struggle to understand potential variability in expenditures that might cause them to spend more than the typical retiree.  In particular, they underestimate how much personal health experience can affect individual spending.These results suggest that misperception of typical spending may not be a primary factor in retirees’ inability to finance out-of-pocket health care costs, but that misperception of the risk of spending above the median is likely an important factor.  We discuss educational,regulatory, and health policy implications of our findings.

Understanding human decomposition has the potential to significantly improve estimations of postmortem intervals (PMI), a key component of forensic investigations (1). The post-mortem interval is one of the most challenging pieces of evidence to obtain in the field of forensic science. In recent years, a novel approach for PMI calculations has emerged through the use of a microbial clock, an estimation tool based on microbial community data (2). This method, based on predictable patterns in microbial community progression, demonstrates that microbes provide an accurate clock starting at death and relies on the ecological changes in the microbial communities in the body and surrounding environment. However, there is not much known about how interactions between soil and the host microbiome influence PMI estimation. In the current study, we utilized specific pathogen free (SPF) and germ-free (GF) mice buried in non- sterile and sterile graves to identify sources of variability in microbial community progression. Intestinal and soil contents were collected over the course of a 21-day decomposition period and bacterial communities were identified by 16S sequencing. We found that GF mice remained sterile over the study period, regardless of soil sterility. In contrast, soil sterility did have an impact on microbial community dynamics in SPF mice. The data demonstrates that microbial communities at the time of death influence the entry of environmental microbes and microbial progression. Together, these results suggest that differences in the host microbiome at the time of death can significantly impact the predictive power of microbial succession in calculating PMI and should be taken into consideration when developing future models.