There is global concern over fish population declines, particularly for economically and culturally valuable salmonid species. Chinook salmon (Oncorhynchus tshawytscha), the largest in the genus Oncorhynchus, are anadromous salmonids native to the United States Pacific Northwest (PNW). Chinook salmon have declined in abundance and several populations are listed as threatened under the Endangered Species Act. Climate change is projected to have a further, significant impact on Chinook salmon populations, as water reservoir storage decreases, reducing river flows and increasing water temperatures. Sub-optimal water temperature has long been recognized as a key factor influencing disease progression in salmonids, by altering the nature or course of the immune response and increasing infectious agent abundance or virulence, to enhance the likelihood of disease progression. While many opportunistic pathogens may be endemic in a watershed, a disease often only manifests when other factors, such as poor environmental conditions, first compromise the host. Flavobacterium columnare, for example, is a re-emerging bacterial pathogen, especially during summer and early fall, and one of the etiological agents of columnaris disease. During the last two decades, columnaris has become one of the most serious threats to nearly all freshwater fishes including anadromous salmonids. Mitigating the effects of fish diseases requires a thorough understanding of the processes that drive host-pathogen interactions under various environmental conditions. There is a paucity of information on whether the outcomes of infections under various environmental conditions are caused by the environment’s effects on the host, the pathogen, or on both organisms. To address this knowledge gap, the objective of my dissertation was to investigate the context-specific dynamics associated with infectious agents. I used juvenile fall-run Chinook salmon, a species of conservation concern in the California Central Valley, as a sentinel for other runs of Chinook salmon listed as threatened or endangered. The overarching hypothesis of this dissertation was that the host physiological response is associated with the host immune- and stress-response, infection burden, and external stressors (e.g., temperature) that also mediate host-pathogen dynamics. The emphasis of my dissertation was on evaluating host-related factors that influence the outcome of pathogen infections for juvenile Chinook salmon, including tissue damage, the host immune response to prevent, control and eliminate pathogens, and detrimental consequences caused by inflammation, during warming. Many tools have been developed to better understand disease progression mechanisms, which could indicate organismal health status. Insight into which aspects of immunity are recruited can be gained by measuring the direction and magnitude of transcriptional changes in host immune gene regulation and can be paired with the measurement of infection severities in the same tissues (Chapter 1). I examined salmon survival following challenge with F. columnare at two temperatures (14 °C and 18 °C), F. columnare abundance, and transcript abundance of pro-inflammatory, acute phase proteins, anti-inflammatory cytokines, immunoglobulins, and stress-related genes in gills over 16 days. I found that with a temperature rise of 4 °C, F. columnare abundance and prevalence increased. Additionally, data emphasize the significance of timely and balanced immune responses in challenged Chinook salmon. I demonstrated that immune activation at elevated water temperatures that Chinook salmon experience during outmigration (18 °C) appeared to elicit a strong response against infection, as demonstrated by higher transcription levels of immune-related genes. The strong immune response, however, did not result in increased salmon survival, as mortality rates were significantly higher in fish challenged with F. columnare at 18 °C compared to those kept at a cooler temperature, 14 °C. Sub-lethal behavioral effects caused by bacterial infections can have long-lasting repercussions on fish ecological fitness through effects on predator avoidance and foraging success. I, therefore, conducted a study to determine whether F. columnare infection can alter the behavior of juvenile fall-run Chinook salmon. Specifically, I quantified locomotion and anxiety-like behavior. I utilized the open field test to assess locomotion followed by a novel object approach test to quantify anxiety. Additionally, I assessed F. columnare abundance in gills, and changes in expression of genes associated with behavior, in the brain tissue (Chapter 2). The results of this study indicated that: first, whereas challenged fish were less receptive to the presence of the novel object, control fish exhibited a range of behavioral responses. Second, there were significant differences in F. columnare abundance among challenged fish over time, with the pathogen abundance rapidly increasing but also showing potential for recovery over time. Lastly, F. columnare challenge elicited differential expression of behavior-related genes as the F. columnare infection progressed. Lastly, research on host-environment-agent interactions has focused on single pathogen studies. In many systems, however, salmon are exposed to a variety of pathogens, and these multiple exposures may synergize and not result in simple additive effects on the host. Therefore, I determined the prevalence and abundance of pathogens in gill and kidney tissues of fish deployed in the Sacramento River (CA, USA). I further determined consequences of pathogen exposure on salmon histological and physiological responses: expression of immune, stress, and development-related genes (Chapter 3). I found that fall-run Chinook salmon were exposed to multiple potential pathogens when out-migrating from the Sacramento River, with Ceratonova shasta and Parvicapsula minibicornis being the two dominant pathogens. I noted that the presence of specific pathogens in fish tissue does not always imply disease establishment and progression, especially for pathogens that are ubiquitous in the aquatic environment. I detected upregulation of immune and stress-related genes in gills before they were detected in kidneys, suggesting that gill biopsy may be useful for early-warning studies on what could potentially be a non-lethal target tissue. Furthermore, expression of investigated genes was altered following deployment; these responses could potentially represent irreversible impacts on salmon, thus demonstrating the threat of pathogen exposure and disease progression to the outmigrating Chinook salmon population. My dissertation indicates that infectious diseases will likely remain and worsen as a conservation issue as climate change continues to reshape host-pathogen dynamics and will significantly influence fish physiology, behavior, and survival. Combining nonlethal sampling with molecular genetic-based identification techniques opens a wide range of possibilities for creative study plans that could potentially address the true complexity of the host-pathogen dynamic in a warming world. Predicting the trajectory of these dynamics will require the inclusion of greater knowledge and tools (e.g., molecular techniques, histopathology, and behavior testing) outlined in this dissertation and other studies. This dissertation research, however, provides a foundation for future avenues of research investigating host-environment-agent interactions and offers crucial knowledge for future conservation efforts.

This dissertation explores subcellular, organismal and community level effects induced by pesticides of concern in agricultural surface water, and evaluates the use of molecular methods; i.e., environmental DNA (eDNA) metabarcoding, in watershed-wide, multitrophic assessments of freshwater biodiversity. I sought to characterize the impacts of chemical disturbance events occurring at multiple levels of biological organization in anthropogenically impacted freshwater ecosystems. I first evaluated the lethality and sublethal effects of two pesticides: imidacloprid (IMI) and chlorantraniliprole (CHL), as single compounds and binary mixtures, on invertebrates (Daphnia magna) and fish (Pimephales promelas). To explore the effects of complex mixtures, I also conducted exposures as described above on contaminated surface water samples collected near agricultural fields associated with the Salinas River Watershed CA (USA). Analytical chemistry data from surface water samples showed chemicals of emerging concern as common analytes at levels expected to cause detrimental effects on aquatic life. I measured acute toxicity in invertebrates exposed to field-collected surface water, and fish exposed to these water samples had significant changes in expression of genes (RT-qPCR) involved with detoxification and neuromuscular function. Exposure of fish to single compounds or binary mixtures of IMI and CHL led to increased relative gene expression of ryanodine receptors (RyR) in fish. Furthermore, IMI targeted the postsynaptic nicotinic acetylcholine receptor (nAChR) in aquatic invertebrates and CHL caused overactivation of RyR in invertebrates and fish. Overall, high levels of invertebrate toxicity and impacts to neuromuscular health in fish are occurring, and pesticides of emerging concern result in detrimental effects in both invertebrates and fish. Based on these findings, I examined behavioral endpoints in the fathead minnow (Pimephales promelas) after the surface water exposures outlined above. I detected differences in both light-induced startle responses and average total distance moved (mm/s), as well as the duration and/or frequency of cruising, bursting and freezing endpoints. These behaviors directly relate to factors influencing survival, feeding and growth, as well as potential for predator avoidance, and thus changes induced by chemical exposure contribute to ecological risk. I detected sublethal and environmentally relevant effects from exposure to contaminated surface waters, which would likely be missed in standard toxicology assessments based on mortality, illustrating the importance of incorporating sublethal endpoints in risk assessments. I then examined behavioral effects of exposure to contaminated surface water before and after a disturbance event (a “first flush” rainstorm at the end of a dry period). I postulated that the swimming behavior of D. magna would be a sensitive bioindicator of exposure to environmentally relevant concentrations of pesticides of concern (IMI and CHL) under laboratory conditions as well as within complex mixtures in contaminated surface waters. I determined that average total distance moved is a sensitive endpoint for pesticide exposure. Daphnia magna response to light stimulus was the most sensitive endpoint measured. In exposures conducted before the first flush event, I detected strong dose-response patterns, with exposed organisms showing a significantly reduced response compared to controls. After first flush, I measured hypoactivity for all sites. I detected different response patterns to light stimulus for each site tested: negative dose-response, non-monotonic, and positive dose-response patterns, with significantly different responses from controls at all concentrations tested. Having determined sublethal and toxic effects from organismal exposure to water samples collected from the Salinas River Watershed, I sought to evaluate how aquatic biodiversity may be distributed across a chemically impacted watershed, and how diversity estimates obtained from both eDNA metabarcoding and morphological identification would correlate with biotic index scores. I collected eDNA from sediment at sites throughout the Salinas River Watershed, across a range of habitat qualities, and compared the resulting taxonomy with morphological data from a subset of high-diversity sites. I detected sensitive invertebrate taxa (Ephemeroptera, Plecoptera, Trichoptera; EPT) from morphology and eDNA, with significant overlap (> 76.67%) between methods, but some taxa were missing from the sequence database, highlighting the importance of taxonomic database development. Sequencing detected more benthic macroinvertebrate taxa than morphology when compared at the genus and species levels of taxonomic resolution. Metabarcoding of sampled eDNA detected rare species of concern and invasive species. Impacted sites contained greater numbers of species known to be tolerant to poor water quality, whereas I only detected several sensitive EPT taxa from least impacted reference sites. Hydrologic distance (waterbody) and biotic index score both accounted for > 27% of the dissimilarity in taxa measured between sites. These findings suggest that biotic indices obtained from eDNA metabarcoding data can be effectively incorporated into watershed-wide, multitrophic assessments of freshwater biodiversity. Freshwater ecosystems in urban and agriculturally developed watersheds are simultaneously exposed to chemical mixtures often include new and emerging contaminants of concern, for which toxicological data may be limited. As the complexity of mixtures increases, non-targeted, effect-based evaluations become necessary for determining potential detrimental outcomes. Through my dissertation work I demonstrate that subcellular, organismal and community level effects are induced by pesticides of concern present in surface water.

Agricultural and urban contaminants enter aquatic environments at concentrations that can affect a variety of sublethal endpoints, including organismal behavior, which can in turn lead to impacts at the population level. The San Francisco Bay Delta (SFBD) is home to multiple threatened aquatic species, such as Delta smelt (Hypomesus transpacificus) and Longfin smelt (Spirinchus thaleichthys), with contaminant exposure likely playing a role in widespread population declines. Pesticides induce hyperactive or hypoactive states in these species, though little is known of the extent of hyperactivity that could be elicited by exposure to neurotoxic compounds. My study used pentylenetetrazole (PTZ), a γ-aminobutyrate (GABA) receptor antagonist, to explore hyperactive behavior in Delta smelt and Longfin smelt. I evaluated induced and spontaneous movement in light and dark conditions following exposure to increasing concentrations of PTZ. Delta smelt and Longfin smelt exposed to PTZ experienced changes in behaviors reflective of induced hyperactivity, including distance moved and swimming velocity, as well as spontaneous hyperactivity, such as time and frequency spent freezing, bursting, or entering the center of the arena (anti-thigmotaxis). The maximum hyperactivity for Delta smelt larvae was recorded following exposure to 8mM PTZ and for Longfin smelt larvae to 4mM PTZ. Together, this information not only confirms that PTZ could be used as a positive control in future behavioral toxicology studies for hyperactivity, but also provides for a better understanding of hyperactive behavior in these species of ecological concern.

Pathogen surveillance must be part of any population supplementation or reintroduction program for the conservation of threatened and endangered species. The unintended transmission of pathogens can have devastating effects on these already at-risk populations or the natural ecosystem at large. In the San Francisco Estuary (estuary), abundance of the endemic Delta Smelt (Hypomesus transpacificus) has declined to the point where regulatory managers are preparing to augment the wild population using fish propagated in a hatchery to prevent species extinction. Although disease is not an overt cause of population decline, comprehensive pathogen presence and prevalence data are lacking. Here, we performed a pilot study that applied molecular assays originally developed in salmonids to assess the presence of a wide variety of pathogens in the gill tissue of cultured and wild Delta Smelt—as well as cultured fish—deployed in enclosures in the estuary. We found the assays to be highly sensitive, and observed positive detections of a single pathogen, Ichthyophthirius multifiliis, in 13% of cultured Delta Smelt. We also detected ten other pathogens at very low levels in cultured, enclosure-deployed, and wild Delta Smelt that likely represent the ambient pathogen composition in the estuary (as opposed to actual infection). Our results corroborate previous work that cultured Delta Smelt do not appear to present a high risk for pathogen transmission during population supplementation or reintroduction. However, the molecular pathogen screening assays tested here have great utility as an early warning system indicator of when further diagnostic testing might be necessary to limit the extent and frequency of disease outbreaks; their utility will be further increased once they are customized for Delta Smelt.