Environmental DNA Plumes: Linking Fish Farm eDNA to Microbial Communities and Novel Detection of Transgenic eDNA

Finfish aquaculture has been on a steady rise, and to match human consumption an increase of open water fish farming is inevitable; however, the impacts of rearing high densities of fish on the surrounding ecosystem remains unclear. Transgenic fish have begun to be implemented in aquaculture to improve traits such as growth rate and feeding efficiency. However, concerns about the potential ecological impact if escaped transgenic organisms are diverse and widespread. Here we characterize the eDNA “plume” from an open water Oncorhynchus tshawystcha farm, and from a transgenic Oncorhynchus kistuch rearing facility. We utilize eDNA as a biomarker of sloughed Chinook salmon DNA from the farm and test for farm effects on bacterial community changes. We found evidence of an overall seasonal effect on eDNA concentration and localized distance effects relative to the farm in the fall. Our BC analyses showed strong seasonal effects as well as evidence of a distance (from the farm) on BC diversity. Despite the well-mixed characteristics of the sampled bay our findings indicate a radial effect of the fish farm plume on the surrounding waters. We also designed a transgene-specific assay to detect transgenic Coho salmon without interference from the wild-type genome and establish the range of detection from an effluent pipe. Our transgene-specific assay detected the growth hormone construct from environmental samples to 10 m from the effluent pipe, as well as two samples 150 m away and 1300m away from the effluent pipe, detecting extremely low traces of transgene DNA copies. This spatial inconsistency in transgenic eDNA detection may be due to sloughed organic matter accumulating, rather then breaking down into a homogenous mixture in marine water. This work establishes how eDNA can be used as a valuable tool for marine surveillance, providing data on the distribution of finfish DNA from a point source and identifying ecological impacts on the surrounding aquatic environment

Spatial and Temporal Microbial Patterns in a Tropical Macrotidal Estuary Subject to Urbanization

Darwin Harbour in northern Australia is an estuary in the wet-dry tropics subject to increasing urbanization with localized water quality degradation due to increased nutrient loads from urban runoff and treated sewage effluent. Tropical estuaries are poorly studied compared to temperate systems and little is known about the microbial community-level response to nutrients. We aimed to examine the spatial and temporal patterns of the bacterial community and its association with abiotic factors. Since Darwin Harbour is macrotidal with strong seasonal patterns and mixing, we sought to determine if a human impact signal was discernible in the microbiota despite the strong hydrodynamic forces. Adopting a single impact–double reference design, we investigated the bacterial community using next-generation sequencing of the 16S rRNA gene from water and sediment from reference creeks and creeks affected by effluent and urban runoff. Samples were collected over two years during neap and spring tides, in the dry and wet seasons. Temporal drivers, namely seasons and tides had the strongest relationship to the water microbiota, reflecting the macrotidal nature of the estuary and its location in the wet-dry tropics. The neap-tide water microbiota provided the clearest spatial resolution while the sediment microbiota reflected current and past water conditions. Differences in patterns of the microbiota between different parts of the harbor reflected the harbor's complex hydrodynamics and bathymetry. Despite these variations, a microbial signature was discernible relating to specific effluent sources and urban runoff, and the composite of nutrient levels accounted for the major part of the explained variation in the microbiota followed by salinity. Our results confirm an overall good water quality but they also reflect the extent of some hypereutrophic areas. Our results show that the microbiota is a sensitive indicator to assess ecosystem health even in this dynamic and complex ecosystem

Role of Vegetated Buffer Zones for Mitigating Wetland Pesticide Contamination and Protecting Aquatic Invertebrate Communities in Northern Prairie Wetlands

Prairie Pothole Region (PPR) wetlands are unique resources that provide a number of ecosystem services. However, the majority of these wetlands have been drained or otherwise degraded due to agricultural activities. Wetlands in the PPR are frequently contaminated by agrochemicals from surrounding agriculture, which has been previously demonstrated to have negative impacts on wetland ecology. Vegetation buffers have been proven to be effective in mitigating pesticide and nutrient contamination of water bodies, but have yet to be fully researched in their efficacy in protecting PPR wetlands. Here I examined how multiple agricultural stressors impact PPR wetland health, and whether natural wetland vegetation or producer-implemented perennial plantings are effective buffers, able to mitigate some of the negative effects of agriculture to wetlands. Measurements of pesticides, nutrients, other water quality parameters, in addition to aquatic invertebrate community endpoints were used to comprehensively evaluate the health of PPR wetlands. Pesticide contamination was widespread, with 59 of the 60 wetlands sampled in 2018 and 2019 containing one or more pesticides in a single growing season. Natural wetland vegetation and the degree of its disturbance from agricultural activities did not have a significant effect on pesticide concentrations in wetlands, although this disturbance did influence the aquatic invertebrate community. Wider and less disturbed wetland vegetation zones were associated with greater macroinvertebrate richness (p = 0.031) and greater abundance of Odonata (p = 0.001). Aspects of water quality were significant predictors of multiple aquatic invertebrate community indices. The occurrence of cyanobacteria blooms as well as increased total nitrogen (TN) were associated with declines in Shannon’s diversity (Cyanobacteria: p = 0.001 and TN: p = 0.016) and Shannon’s Evenness (Cyanobacteria: p = 0.002 and TN: p = 0.001) as well as increases in Berger-Parker Dominance (Cyanobacteria: p = 0.004 and TN: p = 0.001). The Pesticide Toxicity Index (PTIs) calculated for each wetland was associated with changes to the aquatic invertebrate community including a decline in total and relative insect abundance (p = 0.016 and p < 0.001) and an increase in relative snail abundance (p = 0.005). Higher PTIs were also associated with a shift in relative abundance of different functional feeding groups (p = 0.017). This PTI associated shift in taxa and functional feeding groups likely has greater implications for ecosystem function including the many wildlife species that depend on aquatic insects for food. Perennial buffers are considered an important management tool to reduce the negative impacts of agriculture on surface waters. Perennial vegetated buffers recently planted under conservation incentive programs were evaluated for their efficacy in mitigating pesticide and nutrient runoff and protecting wetland health. Wetlands that were fully surrounded by perennial buffers and/or other natural vegetation contained significantly lower concentrations of pesticides (p = 0.001), lower PTIs (p < 0.001), and total phosphorus (p = 0.005). However, the presence of perennial buffers alone did not have a significant effect on pesticide or nutrient detections, and even those wetlands that were fully surrounded by perennial buffers or additional natural vegetation all contained some detectable pesticide contamination. The presence of perennial buffers was significantly associated with greater abundances of macroinvertebrates (p = 0.001), zooplankton (p = 0.005 ), and insects (p = 0.039) which may benefit the many wildlife species that depend on wetland invertebrate productivity for food. This study establishes a framework for using wetland invertebrate communities as an integrative biomonitoring tool for assessing effects of complex agricultural stressors to PPR wetlands. The results from this study demonstrate negative effects of multiple agricultural stressors on wetland health, as measured by changes in the aquatic invertebrate community. Findings here suggest that leaving or planting wetland vegetation around PPR wetlands could increase community richness and abundance of beneficial insects, but is not sufficient for protecting wetlands from pesticide contamination. However, surrounding wetlands with perennial vegetation plantings in addition to other natural vegetation could be an effective method for reducing pesticide and nutrient contamination of wetlands and increasing the abundance and diversity of aquatic invertebrates, which are an important food source for many wildlife species. These findings may help guide producers and land managers motivated to improve wetland health and ecosystem services in prairie agricultural landscapes

The utility of marine neogastropod Californiconus californicus as a model system for investigation of the venom microbiome

The primary question of my dissertation is, “Does venom possess a microbiome specific to it as an ecosystem, and why?” Given the limited amount of literature on venom microbiomes, I selected the California Cone Snail, Californiconus californicus as a proposed, wild model system for studying venom-microbe interactions in the process of investigating hypothesized microbial interactions with host venom. First, I present a data-driven approach for how sampling sites of venomous animals of interest can be selected in conjunction with the current trend to rely on anecdotal information. This work integrates curated museum collections, crowd-sourced data through digital mediums, knowledge through scientific literature, and personal research. This segment delves deeper into our understanding of C. californicus across space and time, dating back to the Pleistocene. We identify relationships between shell morphology and temperature, contributing foundational knowledge for this species and prospective context of venom microbiome coevolution. Second, I characterize the seawater and sediment coastal microbial ecology of the initial known sampling site (Puerto Nuevo, Mexico) in which this species is commonly found. We sampled several sites along a gradient of exposure to urbanization (0.45 km) and characterized the core microbial communities for archaea, bacteria, and microbial eukaryotes using 16S and 18S amplicon sequencing. While only representing one time point and location, our experimental design allows us to demonstrate consistency in the literature in that we identify functionally relevant microbial taxa specific to different environmental types and distance. This work contributes as a preliminary example for the determination of how and where microbes in the venom may be sourced from the wild. Third, I outline the main findings of the venom microbiome for C. californicus. Model organisms used today are common, simplified points of reference for downstream application. The California Cone Snail is a commonly found neogastropod along the California-Baja coast by the 100s. It can be cultured and maintained in a laboratory, acting well for experiments in the wild and in vivo or in vitro. We sampled C. californicus for three major sites for geographical variation: Puerto Nuevo, San Diego, and Monterey. We sampled summer, winter, and summer again, as well as three consecutive days in Puerto Nuevo for temporal variation. We sampled adult and eggs to compare microbial communities across life stage. We then compared venom microbial communities in a lab setting by testing for different hydrostatic pressures, axenic conditions, and exposure to prey. In summary, we find a specific microbial community (16S and 18S) found in and along the venom gland when compared to other environments, tissues, and conditions. Finally, I tie extensions of science outreach together with scientific practice through communication, education, policy, and the first venom-microbe consortium. These initiatives act as proof-of-concept for strengths in democratically practiced open-source, interdisciplinary research through inclusion across demographics and educational and professional stages