TITLE: updated version- Lake biofilms sampling for both downstream DNA analysis  and microscopic counts 
AUTHORS: Frederic Rimet, Rainer Kurmayer, Nico Salmaso, Camilla Capelli, Cecile Chardon, Marine Vautier, Julie Gueguen, Agnès Bouchez, Isabelle Domaizon

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block"><div class = "justify" style = "text-align:left">The objective of this protocol is to provide a reliable and replicable method for the sampling of lake micro-phytobenthos and associated microbes in biofilms, to be used in both downstream DNA analysis and algal microscopic counts. </div></div><div class = "text-block"><div class = "justify" style = "text-align:left">The field protocol is optimized for routine sampling and is in agreement with CEN guidance (NF EN 13946) and CEN technical report (CEN/TR 17245) for the analysis of benthic diatoms from rivers and lakes. The application proposed here in the context of EcoAlpsWater aims at comparing DNA inventories to traditional inventories (microscopy). </div></div><div class = "text-block"><div class = "justify" style = "text-align:left">The sampling strategy has been designed to assess localized hotspots pollutions/perturbations impacts on the shoreline, taking into account shoreline’s heterogeneity.</div></div><div class = "text-block"><div class = "justify" style = "text-align:left">This protocol is part of the deliverables provided by the WP1 of the Eco-AlpsWater project. All members of the EcoAlpsWater consortium have contributed to the optimization of this protocol. </div></div><div class = "text-block"><div class = "justify" style = "text-align:left">The use of microalgae to assess ecological quality of lakes is traditionally based on phytoplankton that allow to evaluate the trophic status. A single depth-integrated water sample from one sampling site above the deepest point of the lake is chosen, and samplings are carried out in the euphotic zone at different seasons. </div></div><div class = "text-block"><div class = "justify" style = "text-align:left">Though not always included as biological quality indicator in lake monitoring, phytobenthos has relevance for lake ecological quality assessment and might be used as a complementary indicator beside phytoplankton in the future (e.g. DeNicola & Kelly 2013).</div></div><div class = "text-block"><div class = "justify" style = "text-align:left">Phytobenthic communities of the lake’s shorelines are generally heterogeneous. The reason for this heterogeneity can be due to many factors, including substrate characteristics as well as anthropogenic factors such as eutrophication, the presence of pollution hotspots, like arrivals of polluted rivers in the lakes or arrivals of wastewaters through drains (e.g. Crossetti et al. 2013, Rimet et al. 2015/2016/2018, Cicek & Yamuc, 2017). But it can also be caused by the nature of the neighboring terrestrial habitats and land use (for instance closed forest vs open fields, or pasture vs steep rock falls).</div></div><div class = "text-block"><div class = "justify" style = "text-align:left">The objective of this protocol is to provide a reliable and replicable method for the sampling of lake micro-phytobenthos and associated microbes biofilms to be used in both downstream DNA analysis and algal microscopic counts. Examples of applications to assess localized hotspots pollutions impact on the shoreline, taking into account shoreline’s heterogeneity, will be provided.</div></div><div class = "text-block"><div class = "justify" style = "text-align:left">The field protocol is based on routine methods used for biofilms sampling and is in agreement with : </div></div><div class = "text-block"><div class = "justify" style = "text-align:left">- CEN 2014. Water quality - NF EN 13946 - Guidance for the routine sampling and preparation of benthic diatoms from rivers and lakes. Afnor, 1-23.</div></div><div class = "text-block"><div class = "justify" style = "text-align:left">- CEN 2018. Water quality - CEN/TR 17245 - Technical report for the routine sampling of benthic diatoms from rivers and lakes adapted for metabarcoding analyses. CEN, 1-8.</div></div></div>

[STEPS]
?. [When & where to sample]
Choice of the sampling season and period-    Phytobenthic communities’ composition are changing along seasons. In large lakes, the major variables explaining these temporal changes are nutrients (and phosphorus especially).-    Moreover, the heterogeneity between the communities present along the shoreline vary from a season to another, and is more important in summer (e.g. Lake Geneva in Rimet et al. 2015). For this reason, samplings must be carried out during summer.-    In general sampling sites free of submerge (and emerge) macrophytes should be selected to exclude shadowing effects on phytobenthic growth which is (ironically) most effective during summer.-    If wind induced strong waves during several days and scoured the biofilms (e.g. storms, days with continuous strong winds), then, you have to wait for 2-3 weeks before collecting the samples, to allow biofilms to restructure. -    Water level fluctuations would also have a significant effect on biofilms community composition. Water level should be stabilized at least over the last 3 weeks and the samples should be taken at 50cm depth from minimum water level (recorded annual data).Choice of the sampling stations (example of application in Eco-AlpsWater monitoring)-    Phytobenthic communities’ composition are changing along seasons. In large lakes, the major variables explaining these temporal changes are nutrients (and phosphorus especially).-    Moreover, the heterogeneity between the communities present along the shoreline vary from a season to another, and is more important in summer (e.g. Lake Geneva in Rimet et al. 2015). For this reason, samplings must be carried out during summer.
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							-    In general sampling sites free of submerge (and emerge) macrophytes should be selected to exclude shadowing effects on phytobenthic growth which is (ironically) most effective during summer.-    If wind induced strong waves during several days and scoured the biofilms (e.g. storms, days with continuous strong winds), then, you have to wait for 2-3 weeks before collecting the samples, to allow biofilms to restructure. 
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							-    Water level fluctuations would also have a significant effect on biofilms community composition. Water level should be stabilized at least over the last 3 weeks and the samples should be taken at 50cm depth from minimum water level (recorded annual data).
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							Choice of the sampling stations (example of application in Eco-AlpsWater monitoring)In the framework of Eco-AlpsWater, at least 10 stations must be sampled in each lake. Usually, samples are taken every 0.5 to 1.5 km along the shoreline (depending on lake size). Since 10 samples are a quite reduced number of samples to cover the entire perimeter of a lake, we propose to choose a small stretch shoreline of the lake. This stretch should be chosen by expert judgment and should present as small heterogeneity in substrate. Sampling site selection strategy (figure 1):-    When shoreline is homogeneous (when natural, or semi-natural), position one sampling site every 0.5 to 1.5 km (depending on lake size).-    When heterogeneity is present along the shoreline, densify the number ofsampling sites, for instance:
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							            *    near the arrival of a river (e.g. in river mouth, and one on each bank)            *    near the arrival of a wastewater treatment plant            *    inside/outside harbors (inside=in the harbor, outside=in the lake)            *    near quarry exploitation (aggregate, stones, sand exploitation)
?. [Biofilms sampling procedure & preservation]
-    Nylon brushes (e.g. toothbrush) must be new    -    At the lab, the tray is cleaned with 10% H2O2, then rinsed with DNA free water and dried    -    Wear gloves throughout the sampling process and change them between different sampling stations
?. [Biofilms sampling procedure & preservation]
If stones are available (figure 2): A demonstration video is available at https://youtu.be/_6Q48nSMjNA Take at least 5 stones (it can be more, depending on stones sizes and biofilms amounts), for a total brushed surface of at least 100 cm2. Stones are taken at 20-50 cm depth from the minimal water level (annual data) in an area of 100 m². The area is at least 2 m wide (a 2 m wide strip corresponds to a 50 m long stretch can be sampled).Let the stones drain for a few minutesFill the bottom of the tray with 50 ml of free DNA waterBrush the stones in the tray. Note: Brush the stones above and on the sides, but not the surface in contact with the ground.The obtained biological material (mixture of biofilms and water) is then sub-sampled:Note: Brush the stones above and on the sides, but not the surface in contact with the ground.    * For DNA samples (figure 2):-    Take the biofilm/water mixture from the tray and fill the tube up to 10 ml -    Complete the tube with absolute ethanol up to 50 ml (add ~40 mL of absolute ethanol)-    Shake to homogenize, label-    The samples are stored at 4°C in the dark for a maximum of 1 month, or can also be frozen at -20 or -80°C for a maximum of 3 months    * For microscopy samples:-    Take a second sub sample (10 mL) of the biofilm/water mixture from the same tray -    Add the appropriate preservative solution for the microscopy analysis according to the case, preservative solution can be Ethanol (70%) or Formaldehyde (2%).Store at ~ +4°C, in the dark.-    If the sample has to be be inspected live under the microscope (in particular for soft algae), a third sub-sample can be kept without any fixation. Store at ~ +4°C, in the dark.
?. [Biofilms sampling procedure & preservation]
If stones are not available: Sample on artificial and hard substrates (figure 3) (e.g. riprap, artificial concrete banks)-    Use a hoe equipped with a net-    Scrap a minimal surface of 100 cm3 at 20-40 cm depth  If artificial and hard substratesare not available, sample on macrophytes-    Use a hoe equipped with a net-    Scrap a minimal surface of 100 cm3 at 20-40 cm depth-    At 20-40 cm depth: squeeze submerged filaments (e.g. Elodea, Potamogeton) or scrap the macrophyte stem (e.g. Typha). At least 20 cm of stem must be scrapped.Important note: if samples are taken on macrophytes, this must be indicated on the sample, and only diatom analyses will be performed on these samples.
?. [Blank sample]
It is recommended to make a blank sample during the sampling for DNA analysis:         -    If number of stations         -    If number of stations > 10, make 1 blank sample every 10 samples approximatelyFill the bottom of a clean tray with 50 ml of free DNA water.Fill a 50 mL sterile Falcon tube up to 10 ml with the water from the tray. Complete the tube with absolute ethanol up to 50 ml (add ~40 mL of absolute ethanol). Shake to homogenize and label. Store the blanks like the other samples (at ~ +4°C in the dark for a maximum of 1 month, or frozen at -20 or -80°C for a maximum of 3 months).
?. [Label standard & sampling field datasheet ]
Sample labeling: “Biofilm”, “River Name”, “Station”, “Date”Accompanying documents: -    field sheets : we propose a field sheet (Table 1) for mesological accompanying data with four subparts (Figure 4):             *    A: Station description            *    B: Sample description            *    C: Shore description            *    D: Land use description-    photographic documentation : if possible, add a photographic documentation of the sampling area