Objective 1: In year 1 (2021), we will survey and quantify the extent of spread in the St. Marys River downstream of the original infestation site (Main Rapids). We will sample nearshore habitat approximately every 1 km from the Main Rapids downstream to DeTour, Michigan in US waters, for a total of approximately 70 longitudinal sample sites. Floating mats will also be recorded and GPS referenced. Three standardized rock scrapes will be collected at each site and the algal sample will be preserved in ethanol and returned to the laboratory where Didymo presence will be verified using microscopy, and algal biomass will be determined using Ash-free-dry-mass protocols (Steinman et al. 2017). Activities and Method(s) In year 1, we will survey and quantify the extent of spread in the St. Marys River downstream of the original infestation site (Main Rapids). We will sample nearshore habitat approximately every 1 km from the Main Rapids downstream to DeTour, Michigan in US waters, for a total of approximately 70 longitudinal sample sites. Floating mats will also be recorded and GPS referenced. Three standardized rock scrapes will be collected at each site and the algal sample will be preserved in ethanol and returned to the laboratory where Didymo presence will be verified using microscopy, and algal biomass will be determined using Ash-free-dry-mass protocols (Steinman et al. 2017). In situ water measurements (e.g., dissolved oxygen, pH, turbidity) will also be recorded using a Hydrolab minisonde 5 multi-probe meter. Water samples will also be collected at each site and analyzed using standard APHA colorimetric methods for color and dissolved nutrients (e.g., phosphorus, nitrogen, calcium) in the Center for Freshwater Research and Education (CFRE) analytical lab using an AQ300 discrete analyzer. In years 1 and 2, we also will survey benthic algal assemblages using combined eDNA and microscopy approaches in about 25 Lake Superior and 25 Lake Michigan tributaries in late May/early June, which likely corresponds to peak bloom timing. These sites will be selected based on surficial geology and resultant water chemistry that likely favors Didymo growth. The eDNA protocols will be adapted from Cary et al. (2014), Keller et al. (2017), and Tiam et al. (2017). These previous studies have used environmental DNA to detect the presence of Didymo in other waters and have demonstrated the importance of verifying samples with microscopy as well. Field samples will be collected using a plankton net (35 micron mesh) and sampling a target volume of 10,000 L (based on flow at each site). Samples will be preserved in 70% ethanol and returned to the lab and frozen until extraction. All eDNA samples will be processed at the CFRE analytical lab using a Didymo-specific eDNA method based on qPCR. Although primers have been identified in previous studies (D602F/D753R; Tiam et al. 2017), we will initially verify detection of Didymo with the primers prior to running all samples. We will also implement QA/QC procedures including negative and positive controls to minimize false positive and false negatives in the testing. Following previous research, we will use the MoBio PowerSoil DNA isolation kit according to manufacturer’s instructions, which has been shown to produce 100% amplification efficiency (Keller et al. 2017). Real-time PCR reactions will then be run on a thermal cycler following methods in Cary et al. (2014). In addition to eDNA samples, three replicate benthic algal samples will be collected at each site by scraping a standardized area of cobble. Samples will be stored in whirlpak bags in 70% ethanol and returned to the lab. All samples will be diluted to a standardized volume, sub-sampled, and then counted using a zooplankton counting tray. This approach is effective at sampling larger volumes to detect Didymo cells because the cells are quite large and easy to identify at lower powers (e.g., 40x). Microscopic identification will be used to compare to and verify positive eDNA results. Didymo sampling will also be paired with water chemistry and habitat data collection (see objective 2) to characterize the suite of environmental conditions in which Didymo is found (or absent from) in Michigan waters. Field crews will implement LSSU’s field decontamination policy after each visit to a waterbody and all field gear will be disinfected between sites using a 5-10% bleach solution. This is key to avoid unintended spread of Didymo to other sites, but also to ensure quality control and avoid contamination of samples from previously sampled sites. All Didymo collected will be preserved in an ethanol or lugol’s solution and maintained in LSSU collections or it will be autoclaved prior to disposing of samples in the waste. This decontamination process will be followed for all sampling associated with this project. Objective 2: To determine the critical thresholds for cell survival and mat formation in Michigan waters, a widespread water chemistry sampling effort will take place in years 1 and 2, and it will be paired with Didymo detection sampling described in Goal and Objective 1 (see above). This study design will enable us to assess the current distribution of cells and blooms in relation to water chemistry across the region. We will sample water chemistry in situ using a Hydrolab minisonde multiprobe meter and grab samples, and we will measure habitat characteristics (e.g., substrate, flow, light) in about 25 Lake Superior and 25 Lake Michigan tributaries as well as throughout the St. Mary's longitudinal profile from the Main Rapids to DeTour (70 sites, 1 each km). Measured parameters at each sampling site will include pH, conductivity, temperature, DO, and turbidity. Grab samples will be processed at CFRE’s analytical labs on an AQ300 discrete analyzer using standard APHA colorimetric methods for color and Activities and Method(s) To determine the critical thresholds for cell survival and mat formation in Michigan waters, a widespread water chemistry sampling effort will take place in years 1 and 2, and it will be paired with Didymo detection sampling described in Goal and Objective 1 (see above). This study design will enable us to assess the current distribution of cells and blooms in relation to water chemistry across the region. We will sample water chemistry in situ using a Hydrolab minisonde multiprobe meter and grab samples, and we will measure habitat characteristics (e.g., substrate, flow, light) in about 25 Lake Superior and 25 Lake Michigan tributaries as well as throughout the St. Mary's longitudinal profile from the Main Rapids to DeTour (70 sites, 1 each km). Measured parameters at each sampling site will include pH, conductivity, temperature, DO, and turbidity. Grab samples will be processed at CFRE’s analytical labs on an AQ300 discrete analyzer using standard APHA colorimetric methods for color and dissolved nutrients (phosphorus, nitrogen, calcium, silica; APHA 2017). This paired sampling effort will help inform our species distribution model based on the conceptual model developed by Cullis et al. 2012. The Didymo Conceptual Model highlighted that water chemistry parameters were key controlling factors for cellular presence (cell survival) and mat presence (mat formation) which defined the habitat window for this alga. In year 2, based on existing literature, combined with presence-absence data related to water chemistry, we will develop a model to predict invasion risk for areas that are susceptible to Didymo blooms. Precise Didymo distribution maps are needed to elaborate a robust approach to understand how water chemistry and other potential controls vary at the local and regional scale. Maps will be produced to 1) detail current distribution resulting from our detection efforts and 2) associated risk level regarding the likelihood of future spread based on suitability of freshwater habitat conditions for bloom development. With results from this effort, combined with results from Objective 1 and 3 (see above and below), we will determine the relative importance of various chemical and physical thresholds and run them in a hierarchical decision tree for predicting Didymo mat presence and severity. This risk assessment, an effective management tool for better predicting the location of nuisance blooms, will identify the top most susceptible sites for the onset of blooms and will strategically inform outreach efforts to educate visitors about Didymo and how to limit its spread. In years 2 and 3, we will give a presentation on Didymo and how to prevent the spread of aquatic invasive species to local community groups including Sault Naturalists, St. Marys River Fishery Task Group, and the local Trout Unlimited chapters. To increase public and stakeholder engagement, in collaboration with the MISN, we will provide interested users, such as anglers, outfitters and state departmental staff, with waterproof monitoring field booklets that will engage citizens in Didymo detection. This citizen science monitoring will help promote early detection and further inform species distribution and presence-absence of blooms in various water bodies of Michigan. Objective 3: Understanding underlying mechanisms of specific water chemistry variables as controls on the growth and persistence of this mat-forming diatom is necessary for effective management. We propose to use outdoor experimental streams to identify the environmental triggers that are controlling Didymo blooms, including mat initiation and proliferation. In year 1, we propose to construct experimental streams adjacent to LSSU’s Center for Freshwater Research and Education, and along the St. Marys River. This location is ideal because it allows replication of river conditions by pulling water directly from the river, but it does so in a controlled environment. We propose to model the experimental streams after Kilroy & Bothwell (2011), which have been used to evaluate Didymo responses to experimental treatments in New Zealand. The experimental streams will have at least 9 stream channels, with channels at least 3 m long and 10 cm wide, and the frame will be constructed to allow changes in gradient. Thus, this design will support maximum versatility by allowing replication of multiple treatments (e.g., 3 reps for 3 treatments) and variation in environmental conditions (e.g., increased flow and gradient). For each experiment, water will be pumped directly from the St. Marys River and collected in a header tank (i.e., reservoir) located above the experimental streams. Water will be gravity fed from the header tank to each channel replicate, and treatment amendments will occur either upstream of the channel via mixing buckets for water quality amendments (e.g, phosphorus) or within the channel for habitat amendments (e.g., canopy/light). Experimental stream facilities have several advantages over other types of aquatic research. Experimental streams allow for true replication of stream units and they provide greater control of potentially confounding variables (such as substrate type, current, light). These first two advantages lead to great statistical power which is often needed when studying inherently variable microbial communities. Additionally, the location of this experimental facility will allow for the use of water that already supports Didymo mat formation. This is critically important since it has been difficult to grow cultures of Didymo in lab settings while ensuring we are using environmentally relevant controls. It is also critical because it allows for Didymo cells to interact with a realistic community of potentially competing species (including bacteria, protozoans, algae, and invert populations) that are found in the natural environment and may Activities and Method(s) Understanding underlying mechanisms of specific water chemistry variables as controls on the growth and persistence of this mat-forming diatom is necessary for effective management. We propose to use outdoor experimental streams to identify the environmental triggers that are controlling Didymo blooms, including mat initiation and proliferation. In year 1, we propose to construct experimental streams adjacent to LSSU’s Center for Freshwater Research and Education, and along the St. Marys River. This location is ideal because it allows replication of river conditions by pulling water directly from the river, but it does so in a controlled environment. We propose to model the experimental streams after Kilroy & Bothwell (2011), which have been used to evaluate Didymo responses to experimental treatments in New Zealand. The experimental streams will have at least 9 stream channels, with channels at least 3 m long and 10 cm wide, and the frame will be constructed to allow changes in gradient. Thus, this design will support maximum versatility by allowing replication of multiple treatments (e.g., 3 reps for 3 treatments) and variation in environmental conditions (e.g., increased flow and gradient). For each experiment, water will be pumped directly from the St. Marys River and collected in a header tank (i.e., reservoir) located above the experimental streams. Water will be gravity fed from the header tank to each channel replicate, and treatment amendments will occur either upstream of the channel via mixing buckets for water quality amendments (e.g, phosphorus) or within the channel for habitat amendments (e.g., canopy/light). Experimental stream facilities have several advantages over other types of aquatic research. Experimental streams allow for true replication of stream units and they provide greater control of potentially confounding variables (such as substrate type, current, light). These first two advantages lead to great statistical power which is often needed when studying inherently variable microbial communities. Additionally, the location of this experimental facility will allow for the use of water that already supports Didymo mat formation. This is critically important since it has been difficult to grow cultures of Didymo in lab settings while ensuring we are using environmentally relevant controls. It is also critical because it allows for Didymo cells to interact with a realistic community of potentially competing species (including bacteria, protozoans, algae, and invert populations) that are found in the natural environment and may have inter-specific synergistic effects. Another benefit of experimental streams in this proposed project is that it allows researchers to measure sensitive response variables such as colonization patterns, cell growth rates, stalk production, and cell health; all of which are challenging in a large-scale field setting. Finally, since Didymo already exists at the location of this facility in the St. Marys River, experiments involving Didymo can be conducted without fear of spreading this invasive species to new areas. This facility and its unique location is projected to function as a research hot-spot for Didymo research for many years after this funding by attracting additional outside funding long-after this funding cycle is over. Once the experimental streams are constructed in year 1, preparation will begin for experiments that will be conducted in spring/summer of years 2 and 3. We propose to conduct a series of experiments where we manipulate potential environmental triggers of blooms including dissolved P concentration, light (via canopy or dissolved organic carbon), and flow. Year 2 experiments will focus on understanding the effect of dissolved nutrients (including ratios) on Didymo cell division and stalk formation. Past research has demonstrated these two response variables do not respond necessarily to the same triggers, and thus this is critical to determine where blooms can occur and to what extent the manipulation of nutrients can control them. Year 3 experiments will then concentrate on the effects of current and light (changes due to canopy and DOC) on growth and colonization. For river systems, current can have both positive (increased access to nutrients, increased spreading events) and negative (substrate scouring) effects. Understanding of these dynamics have important implications for identifying multiple control strategies that may be manipulated in the St. Marys River, which has compensating gates that regulate flow in the river. Exploring the potential role of light related to canopy (using shade cloth) and dissolved organic carbon concentrations could also provide important insights into how global change, including land use development which often reduces canopy cover, and climate change, which is altering DOC concentrations, may mitigate or exacerbate Didymo stalk production. Experiments will primarily be carried out in early May/June, corresponding to natural phenology of peak stalk production in the St. Marys River. Didymo will be “inoculated” into the experimental channels by transplanting small cobbles from the Main or Little Rapids areas of the St. Marys River. To investigate triggers that initiate stalk production, we will collect cobbles prior to the onset of mat growth. To investigate cessation of stalk production, we will collect small cobbles from the river that have high Didymo biomass. Cobbles will be randomly distributed across the stream channels in densities that cover the channel bottom and placed on a thin layer of sand and small gravel, using natural materials from the adjacent river. For treatments evaluating dissolved nutrients as controls on stalk and cell production, initial experiments will be conducted to determine appropriate rates of nutrient drips or fertilizer amendments needed to create desired treatment conditions (e.g., target P thresholds of 5 ug/L to simulate nutrient enrichment) for the duration of the experiment. Response variables measured will include stalk lengths, cell counts, cell division rates (Kilroy and Bothwell 2011), and periphyton biomass (Steinman et al. 2017). Linear mixed effects models will be used to test for statistical differences in response variables among treatments. Findings from these experiments will allow us to understand how environmental variables, such as nutrient conditions and flow, influence the growth of Didymo and provide insights into their competitive strategy to sustain large amounts of biomass in low-nutrient systems. These results can be extrapolated from the experimental streams and applied to targeted areas in the St. Marys River, or other waters experiencing nuisance mat growth, to test innovative techniques to manage and control Didymo.