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Marine environments are under constant pressure from anthropogenic pollution. Chemical pollutants are introduced into the aquatic environment through waste disposal, sewage, land runoff and environmental exploitation (harbours, fisheries, tourism) leading to disastrous effects on the marine wildlife. Developmental malformations, reproduction failure including sex changes and high death rates are commonly observed in aquatic animal populations around the world. Unfortunately, the underlying molecular mechanisms of these pollution effects, in particular for marine invertebrate species, are often unknown. One proposed mechanism through which environmental pollution affects wildlife, is the disruption of nuclear receptors (NRs), ligand-binding transcription factors in animals. Environmental pollutants can directly interact with nuclear receptors, inducing incorrect signals for gene expression and subsequently disrupt developmental and physiological processes. Elucidation of the exact mechanism in invertebrates, however, is sparse due to limited understanding of invertebrate endocrinology and molecular regulatory mechanisms. Here, I have investigated the presence, expression and function of NRs in the Pacific oyster, Crassostrea gigas, and explored their interrelation with known environmental pollutants. Using a suite of molecular techniques and bioinformatics tools I demonstrate that the Pacific oyster possesses a large variety of NR homologs (43 NRs), which display individual expression profiles during embryo/larval development and supposedly fulfil distinct functions in developmental and physiological processes. Functional studies on a small subset of oyster NRs provided evidence for their ability to regulate gene expression, including interactions with DNA, other NRs or small molecules (ligand-binding). Oyster receptors also show a high likeliness to be disrupted by environmental pollutants. Computational docking showed that the retinoid X receptor ortholog, CgRXR, is able to bind and be activated by 9-cis retinoic acid and by the well-known environmental contaminant tributyltin. A potential interaction between tributyltin and the peroxisome proliferator-activated receptor ortholog CgPPAR has also been found. In addition, exposure of oyster embryos to retinoic acids and tributyltin resulted in shell deformations and developmental failure. In contrast, computer modelling of another putative target for pollutants, the retinoic acid receptor ortholog CgRAR, did not indicate interactions with common retinoic acids, supporting a recently developed theory of loss of retinoid binding in molluscan RARs. Sequence analyses revealed six residues in the receptor sequence, which prevent the successful interaction with retinoid ligands. In conclusion, this investigative work aids the understanding of fundamental processes in invertebrates, such as gene expression and endocrinology, as well as further understanding and prediction of effects of environmental pollutants on marine invertebrates.

Primates exhibit a high degree of among-species dietary diversity, which likely exposes them to varying levels of xenobiotic compounds. Many compounds in this category are known to be metabolized by enzymes in the cytochrome P450 superfamily, which has members in all kingdoms of life. Some of these enzymes have been shown to metabolize drugs and xenobiotic toxins and are therefore of great importance for biomedical research and applications. While the pharmacology of P450 enzymes has been studied extensively, our understanding of molecular evolution of this gene family is incomplete, in part because a great variation exists in the number of P450 homologs across genomes. In this dissertation, I aim to show that xenobiotic-metabolizing P450 genes vary in coherent or predictable ways. First, I show that the CYP2C subfamily, responsible for the breakdown of about 20% of pharmaceutical compounds in humans, has undergone a pattern of gene loss in great apes compared to Old World monkeys. Second, I show that P450 subfamilies involved in the largest proportions of pharmacological biotransformation — CYP2C, CYP2D, and CYP3, as well as CYP2E1 — show statistical signals of positive or diversifying selection on the portions of their genes that correspond to substrate-recognition sites. Finally, I illustrate how some of the most dietarily specialized primates in the entire order, the bamboo lemurs of Madagascar (Hapalemur spp. and Prolemur simus), have undergone a clear pattern of genic loss compared to other species of lemur and, especially, in comparison to their closest relative, the ringtail lemur (Lemur catta). Viewed in summary, these findings show that P450 variation exists in comprehensible patterns and that this topic, which has been relatively neglected by many molecular primatologists, deserves future study as more genome assembles and omic resources become available.

Insecticide use is steadily increasing in the United States. Of these, the insecticide Sevin™ has the active ingredient carbaryl, which is a xenobiotic. This is a reverse inhibitor of acetylcholinesterase, which prevents nerve excitation causing paralysis and death in insects. Though insects are the target species, carbaryl has many routes of exposure such as run-off, inhalation, and physical contact making it easy to encounter for non-target species like aquatic life and humans. Carbaryl has been studied within in vitro models to test its effects on zebrafish. The insecticide has already been shown to act on the acetylcholinesterase (AChE) pathway, but it also has potential to act on a second pathway: the aryl hydrocarbon receptor (AhR) pathway. Zebrafish genes and genetic pathways are homologous to humans, allowing for insight on how carbaryl could be affecting humans as well. This study set out to use an in vivo model to show how carbaryl is inducing cytochrome P450 or other AhR pathway gene expression. Gene expression of carbaryl treated embryos were compared to untreated embryos to examine this possible relationship. Cyp1a and cyp1b gene expression was increased with an increase in carbaryl concentration suggesting that carbaryl may be causing induction of the AhR pathway.

The honey bee gut microbiome is essential for protecting this pollinator against abiotic and biotic stressors, including the prevention of harmful gut parasites and pathogens. Previous studies have not only demonstrated a linkage of bee gut dysbiosis to increased immunodeficiencies and pathogen sensitivities, but also report the maladaptation of the gut microbiome in bees exposed to agricultural and apicultural chemistries. There are few techniques available that allow for a simple and reliable analysis of the relative proportions of live and dead gut microbes in bees exposed to these chemistries. Previous techniques for measuring gut symbiont dysbiosis are temporally limited by the digestion and excretion of non-viable, double-stranded DNA (dsDNA) from the host. Here, I will report a propidium monoazide (PMA)-based qPCR technique to quantify the antibiotic- and fungicide-mediated dysbiosis of the bee gut microbiome. Bees fed the antibiotics oxytetracycline and tylosin exhibited a 78% and 82% reduction, respectively, of gut bacteria abundance when compared to untreated bees. Similarly, gut microbes in bees fed chlorothalonil and Fumagilin-B were reduced by 44% and 68%, respectively, compared to untreated bees. These data demonstrate the bee microbiome to be depauperated within 24 h of exposure to agricultural and apicultural chemistries. These data support previous evidence that agrochemical exposures may increase pathogenicity of bee pathogens and gut parasites because of the critical role gut microbiomes play in aiding the host immune system. Fungicides, such as chlorothalonil, are not regulated to the extent of other pesticides and are sprayed during the high activity periods of pollinators when incidental exposure are more likely to occur. This PMA-based qPCR approach, coupled with DNA sequencing, is a useful technology that can rapidly identify changes in abundance and diversity of bee gut symbionts after fungicide or antibiotic exposures. In turn, a PMA-based qPCR approach can assist in the discovery of abiotic and biotic stressors of bee gut symbionts, which is an important step towards reducing the loss of a managed agricultural pollinator. Advisor: Troy D. Anderson

The overall hypothesis of this work is that the physiological microbial stress response could serve as a rapid, sensitive, and mechanistically-based indicator of process upset in biological wastewater treatment systems that receive sporadic shock loads of toxic chemicals. The microbial stress response is a set of conserved and unique biochemical mechanisms that an organism activates or induces under adverse conditions, specifically for the protection of cellular components or the repair of damaged macromolecules. Using traditional immunochemical analysis techniques, the heat shock protein, GroEL, was found to be induced in activated sludge cultures exposed to perturbations of chemicals at all concentrations tested (cadmium, pentachlorophenol, and acetone) or heat stress. As total cadmium concentrations increased above 5 mg/L, there was a significant and consistent increase in effluent volatile suspended solids concentrations from activated sludge sequencing batch reactors relative to unstressed controls, but there was no additional increase in GroEL levels. Stress proteins may serve as sensitive and rapid indicators of mixed liquor toxicity which can adversely impact treatment process performance, but GroEL may not be a good candidate protein for this purpose due to the lack of a dose/response relationship. Additionally, production of stress proteins did not explain the significant deflocculation upsets that were characteristic of many of the industrially-relevant chemicals tested, including pentachlorophenol and cadmium. Although the purpose of stress response mechanisms is protective at the cellular level, the effect may be disruptive at the macroscopic level in engineered bioreactor systems. The goal of the second research phase was to determine whether the bacterial glutathione-gated, electrophile-induced potassium efflux system is responsible for deflocculation observed due to shock loads of toxic electrophilic (thiol reactive) chemicals. The results indicate significant K+ efflux from the activated sludge floc structure to the bulk liquid in response to shock loads of 1-chloro-2,4-dinitrobenzene (CDNB), N-ethylmaleimide (NEM), 2,4-dinitrotoluene (DNT), 1,4-benzoquinone (BQ), and Cd2+ to a bench-scale sequencing batch reactor (SBR) system. In most cases, the stressor chemicals caused significant deflocculation, as measured by an increase in effluent volatile suspended solids (VSS), at concentrations much less than that required to reduce the maximum specific oxygen uptake rate by 50% (IC50). This suggests that electrophile-induced activated sludge deflocculation is caused by a protective bacterial stress mechanism (as hypothesized) and that the upset event may not be detectable by aerobic respirometry. More importantly, the amount of K+ efflux appeared to correlate well with the degree of deflocculation. The transport of other cations including sodium, calcium, magnesium, iron, and aluminum, either to or from the floc structure, was negligible as compared to K+ efflux. In bench-scale SBRs, it was also determined that the K+ efflux occurred immediately (within minutes) after toxin addition and then was followed by an increase in effluent turbidity. K+ efflux and deflocculation responses were similar for bench-scale SBRs and continuous-flow reactor systems, indicating that the periods of elevated exogenous substrate levels typical in SBR systems are not required to activate electrophile-induced K+ efflux or deflocculation. This also suggests that the initial and rapid efflux of K+ immediately following electrophile addition is the factor that leads to deflocculation, not the increase in bulk liquid K+. Sphingomonas capsulata, a bacterium consistent with that found in biological wastewater treatment systems, Escherichia coli K-12, and activated sludge cultures exhibited very similar dynamic efflux/uptake/efflux responses due to the electrophilic stressors, NEM and CDNB, and the thiol reducing agent, dithiothreitol (DTT). The polyether ionophore antibiotic, nigericin, was used to artificially stimulate K+ efflux from S. capsulata and activated sludge cultures. Thus, glutathione-gated K+ efflux (GGKE) activity may cause K+ release from the cytoplasm of activated sludge bacteria into the floc structure and extracellular polymeric substances (EPS) and then diffusion-limited transport into the bulk liquid. It was not possible to resolve the effect of the GGKE system on changes in bulk liquid or floc-associated pH. However, calculations indicate that the localized K+ concentration within the floc structure immediately after chemical stress is consistent with that known to induce floc disruption as a result of KCl addition. Using alkaline phosphatase as a periplasmic marker as well as fluorescent membrane-permeable and impermeable nucleic acid stains, it was determined that a negligible amount of the K+ efflux response was due to lysis of activated sludge microorganisms. The current results are very promising and are the first to suggest that activated sludge upset (i.e. deflocculation) may be caused by a specific protective stress response in bacteria.