Microbiome Dynamics and Pathogen-Driven Impacts in Marine Mollusks: Insights from Oysters and White Abalone

Marine ecosystems are facing various threats, from population declines to diseases that impact their overall health. My dissertation investigates the complex interplay between microbiomes, pathogens, and environmental conditions in three distinct marine organisms: Olympia oysters (Ostrea lurida) in the Puget Sound, Pacific oysters (Crassostrea gigas) in San Diego Bay exposed to OsHV-1 SDB µvar, and white abalone (Haliotis sorenseni) afflicted by Abalone Withering Syndrome. The goal of combining and comparing these systems is to elucidate the crucial role of microbiota in understanding ecosystem and host health, including microbes’ response to environmental variables and their interaction with pathogens. The first chapter focuses on the Olympia oyster, a native species in the Puget Sound that has experienced a substantial population crash. To assess the impact of eelgrass habitat and geographical location on oyster microbiomes, Olympia oysters from a single parental family were deployed at multiple sites, both within and outside eelgrass (Zostera marina) beds. Using 16S rRNA gene amplicon sequencing, I demonstrate that gut-associated bacteria differ significantly from the surrounding environment. Regional differences in gut microbiota are associated with the oyster survival rates at different sites after two months of field exposure. However, eelgrass habitat does not influence microbiome diversity significantly. This research highlights the importance of understanding the specific bacterial dynamics associated with oyster physiology and survival rates in the Puget Sound. In the second chapter, I explore the OsHV-1 SDB µvar, a virus threatening oyster aquaculture globally, with a focus on its microvariant in San Diego Bay. The study investigates the influence of temperature on OsHV-1 SDB µvar infectivity. All microvariants of this virus exhibit limited replication and are unable to induce oyster mortality at lower water temperatures. Through experimental infections of hatchery-raised oysters at temperatures ranging from 15 to 24°C, I found that no oysters died at 15°C but most exposed oysters died above 18°C. The infection took hold faster at 21 and 24°C compared to 18°C. As oysters are often immunocompromised by this viral infection, I also chose to focus on the potential contribution of secondary bacterial infections to the disease. The microbiome of healthy, sick and dead oysters was compared using 16S rRNA gene amplicon sequencing to determine how the microbiome is disrupted by infection and which bacteria may be responsible for further progression of the disease. There is a clear shift in microbiome composition and decreases in evenness following infection with OsHV-1 SDB µvar. The third chapter centers on Abalone Withering Syndrome, characterized by the intracellular parasite Candidatus Xenohaliotis californiensus (CaXc) which disrupts gut morphology leading to starvation and possible death. Investigating the microbiome in endangered white abalone exposed to CaXc over an 11-month period reveals dynamic variations in the fecal microbiome and its distinctiveness from the internal tissue microbiomes. CaXc exposure notably impacts the anterior region of the digestive tract more than the distal tissues and feces, sometimes representing up to 99% relative abundance in the post esophagus samples. This comprehensive analysis incorporates qPCR to quantify pathogen loads over time and feces and in internal tissues. The pathogen is detected after 5 months of exposure and is most abundant in the post-esophagus tissue. The samples with the highest relative abundance of the pathogen were also shotgun sequenced to generate whole genome assemblies of bacteria. This led to the novel assembly of a 90% complete genome for CaXc, which is deposited in a public database. To pair these data with a more holistic understanding of the impact of this pathogen, RNA sequencing data was analyzed for differential gene expression patterns between exposed and unexposed abalone. While functional annotation and prediction was poor on the de novo assembled transcriptome, clear differences exist in gene-level response to CaXc between post esophagus and digestive gland tissue

A halo of reduced dinoflagellate abundances in and around eelgrass beds

Seagrass beds provide a variety of ecosystem services, both within and outside the bounds of the habitat itself. Here we use environmental DNA (eDNA) amplicons to analyze a broad cross-section of taxa from ecological communities in and immediately surrounding eelgrass (Zostera marina). Sampling seawater along transects extending alongshore outward from eelgrass beds, we demonstrate that eDNA provides meter-scale resolution of communities in the field. We evaluate eDNA abundance indices for 13 major phylogenetic groups of marine and estuarine taxa along these transects, finding highly local changes linked with proximity to Z. marina for a diverse group of dinoflagellates, and for no other group of taxa. Eelgrass habitat is consistently associated with dramatic reductions in dinoflagellate abundance both within the contiguous beds and for at least 15 m outside, relative to nearby sites without eelgrass. These results are consistent with the hypothesis that eelgrass-associated communities have allelopathic effects on dinoflagellates, and that these effects can extend in a halo beyond the bounds of the contiguous beds. Because many dinoflagellates are capable of forming harmful algal blooms (HABs) toxic to humans and other animal species, the apparent salutary effect of eelgrass habitat on neighboring waters has important implications for public health as well as shellfish aquaculture and harvesting

Metagenome-assembled genome of withering syndrome causative agent, Candidatus Xenohaliotis californiensis, from endangered white abalone (Haliotis sorenseni).

The genome of Candidatus Xenohaliotis californiensis was assembled from shotgun metagenomic sequencing of experimentally infected white abalone. Ninety-one percent genome completeness was achieved with low contamination. Sequencing this genome provides the opportunity to track pathogen evolution over time, conduct gene expression experiments, and study dynamics between this pathogen and its phage

Variation in Survival and Gut Microbiome Composition of Hatchery-Grown Native Oysters at Various Locations within the Puget Sound.

The Olympia oyster (Ostrea lurida) of the Puget Sound suffered a dramatic population crash, but restoration efforts hope to revive this native species. One overlooked variable in the process of assessing ecosystem health is association of bacteria with marine organisms and the environments they occupy. Oyster microbiomes are known to differ significantly between species, tissue type, and the habitat in which they are found. The goals of this study were to determine the impact of field site and habitat on the oyster microbiome and to identify core oyster-associated bacteria in the Puget Sound. Olympia oysters from one parental family were deployed at four sites in the Puget Sound both inside and outside of eelgrass (Zostera marina) beds. Using 16S rRNA gene amplicon sequencing of the oyster gut, shell, and surrounding seawater and sediment, we demonstrate that gut-associated bacteria are distinct from the surrounding environment and vary by field site. Furthermore, regional differences in the gut microbiota are associated with the survival rates of oysters at each site after 2 months of field exposure. However, habitat type had no influence on microbiome diversity. Further work is needed to identify the specific bacterial dynamics that are associated with oyster physiology and survival rates. IMPORTANCE This is the first exploration of the microbial colonizers of the Olympia oyster, a native oyster species to the West Coast, which is a focus of restoration efforts. The patterns of differential microbial colonization by location reveal microscale characteristics of potential restoration sites which are not typically considered. These microbial dynamics can provide a more holistic perspective on the factors that may influence oyster performance

Host and Water Microbiota Are Differentially Linked to Potential Human Pathogen Accumulation in Oysters.

Oysters play an important role in coastal ecology and are a globally popular seafood source. However, their filter-feeding lifestyle enables coastal pathogens, toxins, and pollutants to accumulate in their tissues, potentially endangering human health. While pathogen concentrations in coastal waters are often linked to environmental conditions and runoff events, these do not always correlate with pathogen concentrations in oysters. Additional factors related to the microbial ecology of pathogenic bacteria and their relationship with oyster hosts likely play a role in accumulation but are poorly understood. In this study, we investigated whether microbial communities in water and oysters were linked to accumulation of Vibrio parahaemolyticus, Vibrio vulnificus, or fecal indicator bacteria. Site-specific environmental conditions significantly influenced microbial communities and potential pathogen concentrations in water. Oyster microbial communities, however, exhibited less variability in microbial community diversity and accumulation of target bacteria overall and were less impacted by environmental differences between sites. Instead, changes in specific microbial taxa in oyster and water samples, particularly in oyster digestive glands, were linked to elevated levels of potential pathogens. For example, increased levels of V. parahaemolyticus were associated with higher relative abundances of cyanobacteria, which could represent an environmental vector for Vibrio spp. transport, and with decreased relative abundance of Mycoplasma and other key members of the oyster digestive gland microbiota. These findings suggest that host and microbial factors, in addition to environmental variables, may influence pathogen accumulation in oysters. IMPORTANCE Bacteria in the marine environment cause thousands of human illnesses annually. Bivalves are a popular seafood source and are important in coastal ecology, but their ability to concentrate pathogens from the water can cause human illness, threatening seafood safety and security. To predict and prevent disease, it is critical to understand what causes pathogenic bacteria to accumulate in bivalves. In this study, we examined how environmental factors and host and water microbial communities were linked to potential human pathogen accumulation in oysters. Oyster microbial communities were more stable than water communities, and both contained the highest concentrations of Vibrio parahaemolyticus at sites with warmer temperatures and lower salinities. High oyster V. parahaemolyticus concentrations corresponded with abundant cyanobacteria, a potential vector for transmission, and a decrease in potentially beneficial oyster microbes. Our study suggests that poorly understood factors, including host and water microbiota, likely play a role in pathogen distribution and pathogen transmission

Temperature and microbe mediated impacts of the San Diego Bay ostreid herpesvirus (OsHV-1) microvariant on juvenile Pacific oysters

The ostreid herpesvirus (OsHV-1) was recently detected in San Diego Bay for the first time in farmed juvenile Pacific oysters (Crassostrea gigas). Due to the virus’ ability to cause mass mortality (50 to 100%), it is important to determine the factors that promote infection as well as the consequences of infection. Here we assess the role of temperature in controlling OsHV-1 induced mortality. Pacific oysters were exposed to the San Diego Bay microvariant of OsHV-1 at 4 different temperatures (15, 18, 21, and 24°C). While OsHV-1 was able to replicate in oyster tissues at all temperatures, it did not induce mortality at 15°C, only at the higher temperatures. Additionally, we examined oyster tissue-associated bacterial response to OsHV-1 infection. As shown previously, bacterial richness increased following OsHV-1 exposure, and then decreased as the oysters became sick and died. Four bacterial taxa linked to the San Diego Bay microvariant infection, including Arcobacter, Vibrio, Amphritea, and Pseudoalteromonas, were the same as those shown for other microvariant infections in other studies from globally distributed oysters, suggesting a similar spectrum of co-infection irrespective of geography and microvariant type. The significant shift in the bacterial community following exposure suggests a weakening of the host defenses as a result of OsHV-1 infection, which potentially leads to adverse opportunistic bacterial infection

Non-targeted tandem mass spectrometry enables the visualization of organic matter chemotype shifts in coastal seawater

Urbanization along coastlines alters marine ecosystems including contributing molecules of anthropogenic origin to the coastal dissolved organic matter (DOM) pool. A broad assessment of the nature and extent of anthropogenic impacts on coastal ecosystems is urgently needed to inform regulatory guidelines and ecosystem management. Recently, non-targeted tandem mass spectrometry approaches are gaining momentum for the analysis of global organic matter chemotypes including a wide array of natural and anthropogenic compounds. In line with these efforts, we developed a non-targeted liquid chromatography tandem mass spectrometry workflow that utilizes advanced data analysis approaches such as feature-based molecular networking and repository-scale spectrum searches. This workflow allows the scalable comparison and mapping of seawater chemotypes from large-scale spatial surveys as well as molecular family level annotation of unknown compounds. As a case study, we visualized organic matter chemotype shifts in coastal environments in northern San Diego, USA, after significant rain fall in winter 2017/2018 and highlight potential anthropogenic impacts. The observed seawater chemotype shifted significantly after a major rain event. Molecular drivers of this shift could be attributed to multiple anthropogenic compounds, including pesticides (Imazapyr and Isoxaben), cleaning products (Benzyl-tetradecyl-dimethylammonium) and chemical additives (Hexa(methoxymethyl)melamine) and potential degradation products. By expanding the search of identified xenobiotics to other public tandem mass spectrometry datasets, we further contextualized their possible origin and show their importance in other ecosystems. The mass spectrometry and data analysis pipelines applied here offer a scalable framework for future molecular mapping and monitoring of marine ecosystems, which will contribute to a deliberate assessment of how chemical pollution impacts our oceans.<br /

Non-targeted tandem mass spectrometry enables the visualization of organic matter chemotype shifts in coastal seawater

Urbanization along coastlines alters marine ecosystems including contributing molecules of anthropogenic origin to the coastal dissolved organic matter (DOM) pool. A broad assessment of the nature and extent of anthropogenic impacts on coastal ecosystems is urgently needed to inform regulatory guidelines and ecosystem management. Recently, non-targeted tandem mass spectrometry approaches are gaining momentum for the analysis of global organic matter composition (chemotypes) including a wide array of natural and anthropogenic compounds. In line with these efforts, we developed a non-targeted liquid chromatography tandem mass spectrometry (LC-MS/MS) workflow that utilizes advanced data analysis approaches such as feature-based molecular networking and repository-scale spectrum searches. This workflow allows the scalable comparison and mapping of seawater chemotypes from large-scale spatial surveys as well as molecular family level annotation of unknown compounds. As a case study, we visualized organic matter chemotype shifts in coastal environments in northern San Diego, USA, after notable rain fall in winter 2017/2018 and highlight potential anthropogenic impacts. The observed seawater chemotype, consisting of 4384 LC-MS/MS features, shifted significantly after a major rain event. Molecular drivers of this shift could be attributed to multiple anthropogenic compounds, including pesticides (Imazapyr and Isoxaben), cleaning products (Benzyl-tetradecyl-dimethylammonium) and chemical additives (Hexa (methoxymethyl)melamine) and potential degradation products. By expanding the search of identified xenobiotics to other public tandem mass spectrometry datasets, we further contextualized their possible origin and show their importance in other ecosystems. The mass spectrometry and data analysis pipelines applied here offer a scalable framework for future molecular mapping and monitoring of marine ecosystems, which will contribute to a deliberate assessment of how chemical pollution impacts our oceans

Host biology, ecology and the environment influence microbial biomass and diversity in 101 marine fish species

Fish are the most diverse and widely distributed vertebrates, yet little is known about the microbial ecology of fishes nor the biological and environmental factors that influence fish microbiota. To identify factors that explain microbial diversity patterns in a geographical subset of marine fish, we analyzed the microbiota (gill tissue, skin mucus, midgut digesta and hindgut digesta) from 101 species of Southern California marine fishes, spanning 22 orders, 55 families and 83 genera, representing ~25% of local marine fish diversity. We compare alpha, beta and gamma diversity while establishing a method to estimate microbial biomass associated with these host surfaces. We show that body site is the strongest driver of microbial diversity while microbial biomass and diversity is lowest in the gill of larger, pelagic fishes. Patterns of phylosymbiosis are observed across the gill, skin and hindgut. In a quantitative synthesis of vertebrate hindguts (569 species), we also show that mammals have the highest gamma diversity when controlling for host species number while fishes have the highest percent of unique microbial taxa. The composite dataset will be useful to vertebrate microbiota researchers and fish biologists interested in microbial ecology, with applications in aquaculture and fisheries management

CALIPSO: A Randomized Controlled Trial of Calfactant for Acute Lung Injury in Pediatric Stem Cell and Oncology Patients

To assess if calfactant reduces mortality among children with leukemia/lymphoma or after hematopoietic cell transplantation (HCT) with pediatric acute respiratory distress syndrome (PARDS), we conducted a multicenter, randomized, placebo-controlled, double-blinded trial in 17 pediatric intensive care units (PICUs) of tertiary care children's hospitals. Patients ages 18 months to 25 years with leukemia/lymphoma or having undergone HCT who required invasive mechanical ventilation for bilateral lung disease with an oxygenation index (OI) > 10 and <37 were studied. Interventions used were intratracheal instillation of either calfactant or air placebo (1 or 2 doses). Forty-three subjects were enrolled between November 2010 and June 2015: 26 assigned to calfactant and 17 to placebo. There were no significant differences in the primary outcome, which was survival to PICU discharge (adjusted hazard ratio of mortality for calfactant versus placebo, 1.78; 95% confidence interval, .53 to 6.05; P = .35), OI, functional outcomes, or ventilator-free days, adjusting for risk strata and Pediatric Risk of Mortality (PRISM) score. Despite the risk-stratified randomization, more allogeneic HCT patients received calfactant (76% and 39%, respectively) due to low recruitment at various sites. This imbalance is important because independent of treatment arm and while adjusting for PRISM score, those with allogeneic HCT had a nonsignificant higher likelihood of death at PICU discharge (adjusted odds ratio, 3.02; 95% confidence interval, .76 to 12.06; P = .12). Overall, 86% of the patients who survived to PICU discharge also were successfully discharged from the hospital. These data do not support the use of calfactant among this high mortality group of pediatric leukemia/lymphoma and/or HCT patients with PARDS to increase survival. In spite of poor enrollment, allogeneic HCT patients with PARDS appeared to be characterized by higher mortality than even other high-risk immunosuppressed groups. Conducting research among these children is challenging but necessary, because survival to PICU discharge usually results in successful discharge to home