Standard sampling and monitoring of fish populations are invasive and time- consuming techniques. The ongoing development of statistical techniques to analyze environmental DNA (eDNA) introduces a possible solution to these challenges. We analyzed and created statistical models for qPCR data obtained from two controlled experiments that were conducted on samples of Coho salmon at the Goldstream Hatchery. The first experiment analyzed was a density experiment whereby varying num- bers of Coho (1, 2, 4, 8, 16, 32 and 65 fish) were placed in separate tanks and eDNA measurements were taken. The second experiment dealt with dilution, whereby three Coho were placed into tanks, removed and eDNA was then sampled at dilution vol- umes of 20kL, 40kL, 80kL, 160kL and 1000kL. Finally, we analyzed a set of field data from several streams in the Pacific North West for the presence of Coho salmon. In the field models, we considered the impact of environmental covariates as well as eDNA concentrations. Our analysis suggests that eDNA concentration can be used as a reliable proxy to estimate Coho biomass. / Graduate / 2021-11-20

Floodplains are among the most productive and biologically diverse freshwater ecosystems on earth. The exchange of nutrients and biota that occurs within these systems during seasonal inundation is essential in maintaining floodplain and river health. Anthropogenic structures, such as weirs, channels, and dams, have altered the natural flood hydrology of floodplain systems minimizing the frequency, strength and duration of flood events. This reduction ultimately leads to the isolation of important floodplain habitat, such as off-channel lakes, from the main channel, decreasing connectivity. Although some studies have examined the productivity of off-channel floodplain lakes in relation to connectivity, most are limited to tropical or highly degraded systems. Northern floodplains are not as well understood, with most of the research limited to the spring, summer, and fall seasons, when waterbodies are free of ice. With research limited to ice free seasons, there is not a full understanding of the year-round processes that occur within these off-channel lake habitats. This knowledge is crucial as the winter season is often when conditions within these habitats are at their most extreme. Such conditions prevent many fish species from permanent settlement; however, no research has been attempted to understand fish presence within these habitats during the winter season. In tropical systems, hypoxia-tolerant species and juveniles utilize these habitats as refuge from intolerant predators, so such habitat may be used similarly in more northern systems. The purpose of this research was to understand the connectivity, limnology and suitability as fish habitat of off-channel floodplain lakes in the Saskatchewan River Delta (SRD), SK, during winter months. I determined the degree of connectivity to the main channel for 26 individual lakes within the SRD by two modern methods: remote sensing imagery, and stable isotopes (δ18O, δ2H). Both of these techniques proved effective at determining connectivity of individual lakes and showed good agreement, with lakes arranged into five connectivity categories using remote sensing imagery. Winter limnological conditions within these lakes were significantly influenced by their degree of connectivity, with lakes that were more connected having characteristics similar to that of the river, with higher levels of dissolved oxygen (DO), nitrates (NO3-NO2), pH, and lower levels of nutrients (TN,TP). Lakes that were less connected were characterized by low levels of DO and nutrients, and high levels of ammonia/ammonium (NH3-NH4), conditions that are not favourable for the survival of many fish species. Some of the more hypoxia-tolerant species found within the SRD appear, however, to use these habitats in the winter. This was supported by detection of fish presence using environmental DNA; five fish species were detected in many of the 26 lakes sampled, but only in lakes with NH3-NH4 levels below 1.77 mg/L and volumes greater than 178000 m3. Together, these analyses suggest the influence of a spring/summer flood pulse on limnology is not limited to the months following a flood event, but rather extends well into the ice-cover season. This knowledge is critical as it points to controls on key processes (e.g. nutrient cycling, provision of fish habitat) during the period when lake conditions are most severe. As a result of human induced climate change, and from increased water demands for agriculture and hydropower, the natural flood pulse is expected to further decrease in size and frequency in large river-wetlands such as the SRD. This will reduce the connection between the floodplain and the main channel, with profound impacts on the SRD ecosystem as a whole. Lakes that currently experience frequent inundation will likely have conditions characteristic of infrequently flooded lakes, with low DO and nutrients and high NH3-NH4. Lakes which currently experience infrequent inundation will likely dry up completely due to decreased water renewal.

Cyanobacterial blooms, such as those recently reported in Great Slave Lake (GSL, NWT), have sparked concern over the occurrence of toxic blooms in the North. This study investigated past and present incidences of cyanobacteria in lakes above latitude 60° N. The abundance of the toxin (microcystin) gene mcyE, as well as genes common to all cyanobacteria (16S rRNA) and bacteria (glnA) were quantified from lake sediment cores using ddPCR. Individual colony isolates from a surface bloom in Yellowknife Bay (GSL) in August 2015 were amplified and identified as non-toxigenic Dolichospermum lemmermannii. Very low levels of microcystin genes were detected through the sediment archives (over ~100-150 yr) of GSL and other lakes, as well as in the plankton of GSL. While recent increases in mcyE were not observed, an increase in the cyanobacterial 16S rRNA and glnA genes was seen through time. In the high Arctic Meretta Lake, gene abundance profiles reflected the effects of past eutrophication and recovery.

Underwater visual censuses (UVCs) are one of the most widely used methods of studying species-rich coral reef fish assemblages. However, a considerable portion of reef fish diversity is missed or underrepresented by these traditional survey techniques. Environmental DNA (eDNA) sampling is an emerging technology that can detect traces of animal DNA from environmental samples, such as water and sediment, potentially including taxa that are missed by UVCs. Here, we assess the complementarity of eDNA to UVCs in surveying coral reef fish communities, particularly for cryptic and cryptobenthic taxa. We further investigate the effect of environmental sample source (water and sediment) and depth (10m and 30m). We conducted UVCs and eDNA sampling in three islands of the Farasan Banks, southern Saudi Arabia. A metabarcoding protocol was applied to environmental samples using a broad-spectrum fish assay targeting 16S mitochondrial DNA. Our eDNA surveys revealed 94 fish species, across 86 genera, 38 families, and 14 orders. Of the species detected by eDNA, 48.9% were also recorded on transects and 60.6% on roving diver surveys. eDNA also detected 6 cryptic, 10 cryptobenthic, and 13 pelagic species. Of these, only one (Eviota guttata) was recorded by UVCs. eDNA species composition was found to be significantly influenced by collection site (islands), and sample source (more species detected from water samples than sediment samples), but not by collection depth (10 versus 30 m depth). Our study provides further evidence that eDNA is an effective tool for the biomonitoring of tropical coral reef fish communities. However, we also stress that improvements are needed in methodology and reference sequence coverage for eDNA to realize its full potential of capturing cryptic and cryptobenthic diversity.

The emergence and rapid spread of antimicrobial resistance (AMR) is a phenomenon that extends beyond clinical settings. AMR has been detected in multiple environmental compartments, including agricultural soils and water bodies impacted by wastewater discharges. The purpose of this research project was to evaluate what factors could influence the environmental persistence of antibiotic resistance genes (ARGs), as well as to identify potential strategies employed by human pathogens to survive in secondary environment outside the host. The first part of this dissertation describes the incidence of the New Delhi metallobeta lactamase gene (blaNDM-1) – an ARG conferring resistance to last resort antibiotics – in the influent of a wastewater treatment facility processing municipal wastewater from Jeddah, Saudi Arabia. Detection of blaNDM-1 was followed by the isolation of a multi-drug resistant strain of E. coli (denoted as strain PI7) at a frequency of ca. 3 x 104 CFU/m3 in the untreated municipal wastewater. Subsequently, we described the decay kinetics of E. coli PI7 in microcosm experiments simulating biological treatment units of wastewater treatment plants. We identified that transition to dormancy is the main strategy prolonging the persistence of E. coli PI7 in the microcosm experiments. Additionally, we observed slower decay of E. coli PI7 and prolonged stability of extracellular DNA in anoxic/anaerobic conditions. In the last chapter of this thesis, the fate of extracellular DNA is further explored. Using as a model Acinetobacter baylyi ADP1, we describe the stimulation of natural transformation frequencies in the presence of chlorination disinfection byproducts (DBPs). Moreover, we demonstrate the ability of BAA to stimulate transformation is associated with its capacity to cause DNA damage via oxidative stress. Overall, this dissertation addresses important knowledge gaps in our current understanding of ARB and extracellular ARG persistence in the environment. The results from this project highlight the importance of retrofitting the existing water treatment process with advance membrane filtration units, and the need to relook into the current disinfection strategies. Wastewater treatment technologies should be assessed for their efficacies in not only inactivating ARB and ARGs, but also whether unintended consequences such as stimulated horizontal gene transfer would occur.

Sensitive and accurate detection methods are critical for monitoring and managing the spread of aquatic invasive species, such as invasive Silver Carp (SC; Hypophthalmichthys molitrix) and Bighead Carp (BH; Hypophthalmichthys nobilis) near the Great Lakes. A new detection tool called environmental DNA (eDNA) sampling, the collection and screening of water samples for the presence of the target species’ DNA, promises improved detection sensitivity compared to conventional surveillance methods. However, the application of eDNA sampling for invasive species management has been challenging due to the potential of false positives, from detecting species’ eDNA in the absence of live organisms. In this dissertation, I study the sources of error and uncertainty in eDNA sampling and develop statistical tools to show how eDNA sampling should be utilized for monitoring and managing invasive SC and BH in the United States. In chapter 2, I investigate the environmental and hydrologic variables, e.g. reverse flow, that may be contributing to positive eDNA sampling results upstream of the electric fish dispersal barrier in the Chicago Area Waterway System (CAWS), where live SC are not expected to be present. I used a beta-binomial regression model, which showed that reverse flow volume across the barrier has a statistically significant positive relationship with the probability of SC eDNA detection upstream of the barrier from 2009 to 2012 while other covariates, such as water temperature, season, chlorophyll concentration, do not. This is a potential alternative explanation for why SC eDNA has been detected upstream of the barrier but intact SC have not. In chapter 3, I develop and parameterize a statistical model to evaluate how changes made to the US Fish and Wildlife Service (USFWS)’s eDNA sampling protocols for invasive BH and SC monitoring from 2013 to 2015 have influenced their sensitivity. The model shows that changes to the protocol have caused the sensitivity to fluctuate. Overall, when assuming that eDNA is randomly distributed, the sensitivity of the current protocol is higher for BH eDNA detection and similar for SC eDNA detection compared to the original protocol used from 2009-2012. When assuming that eDNA is clumped, the sensitivity of the current protocol is slightly higher for BH eDNA detection but worse for SC eDNA detection. In chapter 4, I apply the model developed in chapter 3 to estimate the BH and SC eDNA concentration distributions in two pools of the Illinois River where BH and SC are considered to be present, one pool where they are absent, and upstream of the electric barrier in the CAWS given eDNA sampling data and knowledge of the eDNA sampling protocol used in 2014. The results show that the estimated mean eDNA concentrations in the Illinois River are highest in the invaded pools (La Grange; Marseilles) and are lower in the uninvaded pool (Brandon Road). The estimated eDNA concentrations in the CAWS are much lower compared to the concentrations in the Marseilles pool, which indicates that the few eDNA detections in the CAWS (3% of samples positive for SC and 0.4% samples positive for BH) do not signal the presence of live BH or SC. The model shows that >50% samples positive for BH or SC eDNA are needed to infer AC presence in the CAWS, i.e., that the estimated concentrations are similar to what is found in the Marseilles pool. Finally, in chapter 5, I develop a decision tree model to evaluate the value of information that monitoring provides for making decisions about BH and SC prevention strategies near the Great Lakes. The optimal prevention strategy is dependent on prior beliefs about the expected damage of AC invasion, the probability of invasion, and whether or not BH and SC have already invaded the Great Lakes (which is informed by monitoring). Given no monitoring, the optimal strategy is to stay with the status quo of operating electric barriers in the CAWS for low probabilities of invasion and low expected invasion costs. However, if the probability of invasion is greater than 30% and the cost of invasion is greater than $100 million a year, the optimal strategy changes to installing an additional barrier in the Brandon Road pool. Greater risk-aversion (i.e., aversion to monetary losses) causes less prevention (e.g., status quo instead of additional barriers) to be preferred. Given monitoring, the model shows that monitoring provides value for making this decision, only if the monitoring tool has perfect specificity (false positive rate = 0%).

O Brasil apresenta uma das maiores diversidade de espécies de anfíbios, sendo reconhecidas em torno de 500 espécies endêmicas no país, as quais são encontradas predominantemente em área de Mata Atlântica. O monitoramento da herpetofauna e conhecimento da dinâmica espacial e temporal destas espécies são informações básicas, porém, fundamentais ao desenvolvimento de outras áreas de pesquisa e conservação. Neste trabalho reunimos informações sobre ocorrência e abundância de três espécies típicas de riacho, Cycloramphus boraceiensis, Hylodes asper e Hylodes phyllodes e testamos o uso de DNA ambiental para detecção de comunidades de anuros. As amostragens ocorreram em um transecto de 100 a 115 m em quatro riachos no Núcleo de Picinguaba, localizado no Parque Estadual da Serra do Mar, São Paulo, Brasil. Coletas de abundância e uso do habitat ocorreram mensalmente de janeiro de 2007 a dezembro de 2010 e em meses alternados em 2011. Indivíduos pós-metamórficos foram amostrados por procura visual a montante de cada riacho, verificando-se todos os locais ao longo do leito. A localidade de cada indivíduo ativo e inativo foi mapeada e o uso do ambiente foi caracterizado em relação a seis parâmetros ecológicos. As amostras de DNA ambiental foram coletadas em 16 pontos em Abril de 2015. eDNA metabarcoding foi realizado com primer universal de anfíbios para uma região do gene mitocondrial (12S). Registramos um total de 6335 observações visuais. A abundância das três espécies variou entre e ao longo dos riachos, sendo que apenas a espécie Hylodes phyllodes foi registrada no riacho 2. Houve uma sazonalidade na abundância de Cycloramphus boraceiensis e Hylodes asper, sendo ambas espécies encontradas em maior número na estação chuvosa. As três espécies foram encontradas ativas majoritariamente em rochas úmidas ou molhadas, sem musgo e sem cobertura. Indivíduos inativos de Hylodes asper e H. Phyllodes foram encontrados majoritariamente em folhas secas sem musgo e sem cobertura. Por meio da técnica de eDNA metabarcoding, detectamos nove espécies, compatíveis com a amostragem tradicional. O DNA de espécies com fases do ciclo de vida atreladas aos riachos e com maior constância na amostragem tradicional foi detectado em maior proporção. Nossos estudos demonstraram que os resultados da amostragem tradicional e de eDNA metabarcoding fornecem informações fundamentais e complementares, sendo uma combinação de ambas metodologias potencialmente útil a futuros estudos de ecologia / Brazil ranks as the country with one of the highest amphibian species diversity. Streams in the Atlantic forest of southeastern Brazil have an important availability of microenvironments and harbors a particular richness in amphibian species. Monitoring herpetofauna and knowledge on their spatial and temporal dynamics provide primary information for ecological studies, and are essential to the development of other areas such as conservation biology. In this work we gather information on the occurrence and abundance of three torrent frogs, Cycloramphus boraceiensis, Hylodes asper and Hylodes phyllodes and examine the reliability of eDNA analysis to detect anuran communities. Samplings occurred within a 95 to 115 m transect in four streams in Núcleo Picinguaba, at the Parque Estadual da Serra do Mar, São Paulo, Brazil. Individual encounter number and their habitat were monthly recorded from January 2007 to December 2010 and every two months in 2011. We searched for post-metamorphic individuals while walking upstream for 30-60 min, checking all visually accessible spots in the streambed. The location of each active and inactive individual was mapped and its habitat use was characterized in relation to five ecological parameters. We collected eDNA samples at 16 sites on April, 2015. We used eDNA metabarcoding approach with a universal amphibian primer of a mitochondrial marker (12S) to detect amphibian communities. We recorded a total of 6335 visual observations. The three species abundance varied along and between streams and only Hylodes phyllodes were found in the stream 2. Abundance of C. boraceiensis and H. asper was significantly higher in the wet seasons. The three species were found active mainly in wet rocks, without moss and without cover. Inactive individuals of Hylodes asper and H. phyllodes were found mainly in dry leaves, without moss or cover. Through eDNA metabarcoding, we detected nine species, which were consistent with traditional survey results. DNA of riparian species and species with higher constancy in traditional surveys were detected in higher proportions. Our study showed that traditional survey and DNA metabarcoding results can be complementary and both methodologies can be combined in future ecology studies

<p>Environmental DNA (eDNA) surveys utilize DNA shed from animals in order to detect their presence. Since it was developed, this technique has been applied to numerous species across several taxa. In some cases, it has been shown to be superior to traditional survey methods at detecting rare or cryptic species. It allows for the detection of animals in low numbers and does not require direct capture of an animal. This allows eDNA to be more effective at detecting rare or cryptic species that require high survey effort to find. This often reduces survey costs as many eDNA samples can be collected quickly with little equipment required.</p> <p>The Kirtland’s Snake (<i>Clonophis kirtlandii</i>) is a small Natricine snake endemic to the Midwest. It is a species of conservation concern since it is threatened throughout its range. Due to its cryptic and fossorial lifestyle, it is also a notoriously difficult snake to survey. This has resulted in a poor understanding of Kirtland’s Snake life history and population status. Applying eDNA surveys to this species may increase detection probability, offering a more efficient way to survey for them. </p> <p>In this study, a quantitative PCR (qPCR) assay was designed and tested alongside traditional coverboard surveys. The assay had a limit of detection of 166 copies of Kirtland’s Snake DNA. In crayfish burrow sediment, eDNA was found to be detectable up to 10 days and may persist for up to 25 days. However, only one detection occurred out of 380 field samples. Coverboard surveys revealed temporal and spatial variation in Kirtland’s Snake abundance. More snakes were captured in the spring, during the first field season, and at the south coverboard transects. Kirtland’s Snake abundance was also found to be related to the presence of grass and herbaceous vegetation as well as close proximity to shrubs. Comparing survey methods, coverboards resulted in far better snake detection, suggesting that eDNA does not offer an advantage over traditional survey methods for this species. </p>

<p>The broader aim of this work was to investigate the implementation of metagenomic library construction and sequencing-based approaches, as a basis for gene identification and functional characterization, from a novel thermophilic environment.</p>

<p>The broader aim of this work was to investigate the implementation of metagenomic library construction and sequencing-based approaches, as a basis for gene identification and functional characterization, from a novel thermophilic environment.</p>