This research was funded in part by the Gordon and Betty Moore Foundation Marine Microbiology Initiative, through grant GBMF3801 to J.R.S. and R.S., and an Investigator Award (GBMF3783) to R.S., as well as an Australian Research Council Fellowship (DE160100636) to J.B.R., an award from the Simons Foundation to B.S.L. (594111), and a grant from the Simons Foundation (542395) to R.S. as part of the Principles of Microbial Ecosystems (PriME) Collaborative.

We recommend executing section 1 prior to field experiments to optimize results.
1. Laboratory optimization
NOTE: The volumes described in the optimization procedure are sufficient for a single ISCA (composed of 20 wells).
<ol>
	<li>Preparation of the chemical of interest 
	NOTE: The optimal concentration for each chemoattractant often needs to be determined under laboratory conditions prior to field deployments. The chemical concentration field will decrease in magnitude with distance from the source (ISCA well), which means that the concentration experienced by microorganisms in the environment will be lower than that present inside the device. The optimal concentration to use in the ISCA well depends on the diffusion rate of the chemoattractant. If the concentration in the well is too low (close to the detection limit of the microbes), no positive chemotaxis will be observed. Conversely, if the concentration in the well is too high, the concentration will also be high in the immediate environment and microbial accumulation will occur above the ISCA wells rather than inside. Therefore, dilution series should be carried out in laboratory conditions for each compound in order to determine the optimal concentration for field deployments. Ideally, a concentration range should also be tested in the field to confirm laboratory results.
	<ol>
		<li>Prepare 2.5 L of a suitable medium containing the appropriate salt concentration (e.g., phosphate-buffered saline [PBS] or artificial seawater). Filter the medium with a 0.2 &#181;m filter using a peristaltic or vacuum pump and autoclave.</li>
		<li>Prepare a 10 mM solution of chemoattractant in 1 mL of the sterile medium. Filter the chemoattractant solution with a 0.2 &#181;m syringe filter to remove particles and potential contaminants. 
		NOTE: Ideally, no organic compounds other than the chemoattractant should be present in the final solution.</li>
	</ol>
	</li>
	<li>Concentration range by dilution series
	<ol>
		<li>Dilute the filtered chemoattractant stock solution in series, ranging (for example) from 10 mM to 100 &#181;M. 
		NOTE: At least a 0.7 mL final volume is needed per concentration tested.</li>
	</ol>
	</li>
	<li>Loading the ISCA
	<ol>
		<li>Fill a 1 mL syringe with the filtered chemoattractant and connect it to a 27 G syringe. Holding the ISCA with the port facing upward, slowly inject the substance until a small droplet appears on top of the port. 
		NOTE: 1) Each dilution or substance must be injected with a separate syringe and needle to prevent cross-contamination. 2) This small droplet is important because it ensures that no air bubble is trapped within the port, which could impair the ability of microbes to migrate through the port. 3) It is recommended to fill an entire row per substance or concentration (five wells) to provide an adequate minimal volume for further analyses. 4) One row per ISCA should act as negative control and should be filled with the filtered medium in which the microbes will be suspended. This treatment accounts for the effect of random motility by microbes into the ISCA wells and should be used to normalize the treatments containing a chemoattractant.</li>
	</ol>
	</li>
	<li>Deployment in the laboratory
	<ol>
		<li>Overnight, incubate a 5 mL culture enriched with 1% marine broth (for marine bacteria) or 1% lysogeny broth (LB, for fresh water bacteria). 
		NOTE: Motile bacterial isolates or natural bacterial communities can be used for laboratory deployments.</li>
		<li>Incubate the culture for 12 h at room temperature (RT) and 180 rpm. After 12 h, ensure that the microbial communities are motile by direct observation under a microscope.</li>
		<li>Spin down the culture at 1,500 x g for 10 min and resuspend 1/100 in 150 mL of appropriate medium (e.g., filtered seawater, filtered freshwater).</li>
		<li>Place two small pieces of double-sided adhesive tape on the flat surface of a 200 mL capacity tray (the lids of 1 mL tip boxes have the ideal dimensions for this purpose and can easily be autoclaved). Place one ISCA on top, ensuring that it attaches securely to the tape. Slowly fill the deployment tray with the bacterial solution using a 50 mL serological pipette. 
		NOTE: Fill the tray until the ISCA is submerged under approximately 1&#8211;2 cm of liquid. If using multiple trays, use the same volume across all.</li>
		<li>Leave the ISCA to incubate for 1 h to allow bacterial chemotaxis. After 1 h, remove the medium very gently with a 50 mL serological pipette to minimize turbulent flow.</li>
		<li>Retrieve the ISCA from the deployment tray without touching the upper surface. Use a pipette and disposable wipers to remove remaining liquid on the ISCA surface. 
		NOTE: It is important to avoid touching the ports during this process, as the resulting changes in pressure can remove or add bacteria from the outside environment into the well and thereby bias the bacterial density and composition inside the well.</li>
	</ol>
	</li>
	<li>Retrieval of the samples
	<ol>
		<li>Holding the ISCA with the port facing downward, retrieve the volume of the wells using a sterile 27 G syringe needle attached to a 1 mL syringe. 
		NOTE: Each row (if containing the same substance) can be pooled to provide a working sample of approximately 550 &#181;L. This sample can subsequently be aliquoted into different tubes depending on the required downstream applications.</li>
		<li>Determine the number of bacteria attracted to each chemoattractant concentration by analyzing the samples with flow cytometry12. Choose the concentration of chemoattractant that maximizes chemotaxis for subsequent field deployments.</li>
	</ol>
	</li>
</ol>
2. Preparation for field deployment
NOTE: Preparation of material and construction of the flow-damping enclosure (section 2) must be conducted prior to deployment.
<ol>
	<li>Preparation of materials
	<ol>
		<li>Prepare all materials listed in Table 1. 
		NOTE: Material quantities are provided for one ISCA.</li>
	</ol>
	</li>
	<li>Construction and preparation of the flow-damping enclosure 
	NOTE: The flow-damping enclosure minimizes unwanted turbulences that otherwise prevents the establishment of chemical gradients emanating from the ISCA.
	<ol>
		<li>Cut the pieces for the deployment enclosure with a laser cutter from a 3 mm acrylic sheet. 
		NOTE: The file for the pieces can be found using the following link: &#60;https://figshare.com/articles/Flow_damping_enclosure_for_ISCA_deployments/10630220&#62;.</li>
		<li>Assemble the laser-cut pieces as demonstrated in Figure 2 using acrylic glue. 
		NOTE: Assemble the pieces with care. Holes or misalignment can create leaks upon deployment, which directly impacts data quality.</li>
		<li>Leave the assembled enclosure to dry overnight.</li>
		<li>Wash the enclosure with deionized water.</li>
		<li>Identify potential leakage by pouring deionized water into the enclosure. Fix any potential leaking joints by adding more acrylic glue, then repeat steps 2.2.3&#8211;2.2.5.</li>
		<li>Cut the screw threads into the acrylic piece that will be used to secure the ISCA. This can be achieved using a tap with a diameter and pitch matching the mounting screws.
		<ol>
			<li>First, affix the tap into a tap wrench, then secure the acrylic piece to be tapped in a benchtop vice. For the best results, make sure the acrylic piece is as level as possible. Make sure that the tap is perpendicular to the acrylic piece and start turning the tap wrench (clockwise), applying light pressure to the tap.</li>
			<li>After several full revolutions in the acrylic piece, reverse the rotation of the tap (counterclockwise) for one-quarter of a rotation to clear acrylic from the tap. Repeat the process until the entire depth of the acrylic piece is tapped.</li>
			<li>Finally, remove the tap (turning counterclockwise) and test the threads using a screw.</li>
		</ol>
		</li>
	</ol>
	</li>
</ol>
3. Procedure in the field
<ol>
	<li>Water filtration
	<ol>
		<li>Collect water from the field site when ready to start the experiment. Filter 5 mL of water per ISCA through a 0.2 &#181;m syringe filter (with a 50 mL syringe) into a 50 mL conical centrifuge tube. 
		NOTE: Approximately 3 mL of filtered water are required to fill all the wells of an ISCA; however, it is recommended to 1) filter 5 mL per device to account for losses during the quadruple filtration process, and 2) preserve aliquots of the filtrates as negative controls for both flow cytometry and molecular procedures.</li>
		<li>Filter the filtrate twice through a 0.2 &#181;m hydrophilic GP filter cartridge (using the same one, both times) with a new 50 mL syringe into a new 50 mL conical centrifuge tube. Filter the filtrate through a 0.02 &#181;m syringe filter (with a new 50 mL syringe) into a new 50 mL conical tube. 
		NOTE: This quadruple filtration should remove nearly all microorganisms and particles from the water. Keep the final filtrate away from any source of heat until use. This water will be used to resuspend all chemicals used in the ISCA, and it should be maintained at the same temperature as the water at the deployment site. Convective flows triggered by differences in temperature between the ISCA wells and outside environment may otherwise occur.</li>
		<li>Use aliquots of the filtrate to resuspend all chemoattractants of interest (typically dry) to the desired concentrations in 15 mL conical centrifuge tubes.</li>
		<li>Filter the resuspended chemoattractants through a 0.2 &#181;m syringe filter with a 10 mL syringe into sterile 15 mL conical centrifuge tubes to remove unwanted particles or water-insoluble compounds (if using extracts). 
		NOTE: Filter gently to prevent particles from passing through the filter. It is important to resuspend the chemoattractants in the ultrafiltered water from the field site and not solubilize them into other solutions. Using water from the field site is necessary to (1) obtain the same salt concentration inside the wells as that in the bulk environmental water to prevent density-driven flow, and (2) guarantee that background nutrient levels are equal inside and outside of the well.</li>
	</ol>
	</li>
	<li>ISCA filling
	<ol>
		<li>Perform section 1.3 to fill the ISCA. 
		NOTE: It is recommended to fill one row (five wells) per substance (i.e., three different substances per ISCA and one ultrafiltered seawater control).</li>
	</ol>
	</li>
	<li>Deployment in the field
	<ol>
		<li>Screw the ISCA (Figure 3A) to piece 9 of the enclosure (Figure 2K and Figure 3B). 
		NOTE: The flow-damping enclosure outlined above can contain two ISCAs side-by-side or one ISCA placed at its center.</li>
		<li>Close the enclosure (Figure 3C) and seal it with adhesive tape (Figure 3D). 
		NOTE: Wrinkles must be avoided to ensure a perfect seal. Seal all sides first, then (in a second step) seal the side holes, which will be used to drain water from the enclosure at the end of sampling. Do not seal the top and bottom holes. Do not place the ISCA upside down, as density-driven flow can occur in wells containing chemoattractants, which will bias the number of cells in the wells.</li>
		<li>Because the enclosure must remain steady during deployment, it is recommended to attach it to manmade structures (e.g., pontoon, ladder) using bungee cords. 
		NOTE: The enclosure can be attached to a deployment arm (here, a modified clamp with a perpendicular platform) using bungee cords before immersion in the water. Alternatively, the enclosure can be filled and secured with a small weight on shallow substrates. If deployments are intended in the pelagic ocean, the enclosure can be attached to a net with a buoy on one side and dive weight on the other.</li>
		<li>Submerge the enclosure completely to start filling. While filling, hold the enclosure firmly to prevent excessive water movement inside. Once the level of the water reaches the top of the enclosure, make sure that no air is trapped inside. 
		NOTE: In case some air bubbles are trapped, tilt the enclosure gently with the vent hole facing upward, which will enable the bubbles to escape.</li>
		<li>Once completely full, seal the bottom and top holes with two plugs, which can be made out of silicon or rubber or by sealing the extremities of 20 &#181;L pipette tips (Figure 4). 
		NOTE: This step prevents flow inside the enclosure during sampling.</li>
		<li>Leave the ISCA in place for sampling for 1&#8211;3 h. 
		NOTE: The ideal deployment time is primarily dictated by the temperature of the water and doubling time of the bacterial community. When the water temperature is above 20 &#176;C, it is not recommended to deploy the ISCA for more than 1 h, because cell division can occur in the wells containing chemoattractants after 1.5&#8211;2.0 h. However, optimal deployment time can be tested prior to the ISCA experiment by amending natural communities with the loaded chemicals and quantifying the number of cells through time.</li>
		<li>Remove the enclosure from the water. Place it over a container enabling the water to be drained from the enclosure.</li>
		<li>Remove the upper part of the adhesive tape from the front holes very gently. 
		NOTE: The flow of the water leaving the enclosure must be at a dripping speed. Proceed one hole at a time, from the top of the enclosure to the bottom. It should take approximately 10&#8211;15 min to drain the enclosure completely.</li>
		<li>Once the waterline passes below the top of the ISCA, remove the bottom plug, and drain the rest of the water.</li>
		<li>While the ISCAs are still attached to the enclosure, carefully remove the water trapped on top of the ISCA with a 1 mL pipette.</li>
		<li>Remove the ISCA without touching the upper surface and use a disposable wipe to remove any remaining liquid on the surface. 
		NOTE: It is important not to touch the ports during this process, as the resulting changes in pressure can remove or add bulk bacteria into the well and bias the bacterial counts.</li>
		<li>Retrieve the samples from the ISCA by repeating step 1.5.1.</li>
	</ol>
	</li>
</ol>
4. Downstream applications
NOTE: Volumes are given based on a 550 &#181;L sample (one row of an ISCA).
<ol>
	<li>Fix 100 &#181;L of well contents with glutaraldehyde (2% final concentration) for flow cytometry to quantify chemotaxis to each attractant. 
	NOTE: Store on ice (or at 4 &#176;C) and analyze the samples on the same day. Alternatively, samples can be flash frozen in liquid nitrogen following fixation if analysis is not feasible on the same day. Flow cytometry is the recommended method to quantify the number of cells in the ISCA wells, as it is straightforward, fast, and accurate12.</li>
	<li>Snap freeze 300 &#181;L of well content in liquid nitrogen for subsequent DNA extraction and analysis11. 
	NOTE: Store the samples at -80 &#176;C until analysis.</li>
	<li>Add 90 &#181;L of well contents to 10 &#181;L of TE-glycerol buffer and snap-freeze the samples for single-cell genomics13.</li>
	<li>Spread 10&#8211;20 &#181;L on agar plates containing the desired medium for bacterial isolation.</li>
</ol>

<ol>
	<li>Stocker, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Marine+microbes+see+a+sea+of+gradients.">Marine microbes see a sea of gradients.</a> Science. 338, 628-633 (2012).</li><li>Raina, J. B., Fernandez, V., Lambert, B., Stocker, R., Seymour, J. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+role+of+microbial+motility+and+chemotaxis+in+symbiosis.">The role of microbial motility and chemotaxis in symbiosis.</a> Nature Reviews Microbiology. 17, 284-294 (2019).</li><li>Chet, I., Asketh, P., Mitchell, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Repulsion+of+bacteria+from+marine+surfaces.">Repulsion of bacteria from marine surfaces.</a> Applied Microbiology. 30, 1043-1045 (1975).</li><li>Smriga, S., Fernandez, V. I., Mitchell, J. G., Stocker, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chemotaxis+toward+phytoplankton+drives+organic+matter+partitioning+among+marine+bacteria.">Chemotaxis toward phytoplankton drives organic matter partitioning among marine bacteria.</a> PNAS. 113, 1576-1581 (2016).</li><li>Matilla, M., Krell, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+effect+of+bacterial+chemotaxis+on+host+infection+and+pathogenicity.">The effect of bacterial chemotaxis on host infection and pathogenicity.</a> FEMS Microbiology Reviews. 42, (2018).</li><li>Adler, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chemotaxis+in+bacteria.">Chemotaxis in bacteria.</a> Science. 153, 708-716 (1966).</li><li>Adler, J., Dahl, M. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+method+for+measuring+the+motility+of+bacteria+and+for+comparing+random+and+non-random+motility.">A method for measuring the motility of bacteria and for comparing random and non-random motility.</a> Journal of General Microbiology. 46, 161-173 (1967).</li><li>Ahmed, T., Shimizu, T. S., Stocker, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microfluidics+for+bacterial+chemotaxis.">Microfluidics for bacterial chemotaxis.</a> Integrative Biology. 2, 604-629 (2010).</li><li>Hol, F. J. H., Dekker, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Zooming+in+to+see+the+bigger+picture:+microfluidic+and+nanofabrication+tools+to+study+bacteria.">Zooming in to see the bigger picture: microfluidic and nanofabrication tools to study bacteria.</a> Science. 346, 1251821 (2014).</li><li>Rusconi, R., Garren, M., Stocker, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microfluidics+expanding+the+frontiers+of+microbial+ecology.">Microfluidics expanding the frontiers of microbial ecology.</a> Annual Review of Biophysics. 43, 65-91 (2014).</li><li>Lambert, B. S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+microfluidics-based+in+situ+chemotaxis+assay+to+study+the+behaviour+of+aquatic+microbial+communities.">A microfluidics-based in situ chemotaxis assay to study the behaviour of aquatic microbial communities.</a> Nature Microbiology. 2, 1344-1349 (2017).</li><li>Marie, D., Partensky, F., Jacquet, S., Vaulot, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Enumeration+and+cell+cycle+analysis+of+natural+populations+of+marine+picoplankton+by+flow+cytometry+using+the+nucleic+acid+stain+SYBR+Green+I.">Enumeration and cell cycle analysis of natural populations of marine picoplankton by flow cytometry using the nucleic acid stain SYBR Green I.</a> Applied Environmental Microbiology. 63, 186-193 (1997).</li><li>Rinke, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Obtaining+genomes+from+uncultivated+environmental+microorganisms+using+FACS-based+single-cell+genomics.">Obtaining genomes from uncultivated environmental microorganisms using FACS-based single-cell genomics.</a> Nature Protocols. 9, 1038-1048 (2014).</li><li>Gaworzewska, E. T., Carlile, M. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Positive+chemotaxis+of+Rhizobium+leguminosarum+and+other+bacteria+towards+root+exudates+from+legumes+and+other+plants.">Positive chemotaxis of Rhizobium leguminosarum and other bacteria towards root exudates from legumes and other plants.</a> Microbiology. , (1982).</li><li>Walker, T. S., Bais, H. P., Grotewold, E., Vivanco, J. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Root+exudation+and+rhizosphere+biology.">Root exudation and rhizosphere biology.</a> Plant Physiology. 132, 44-51 (2003).</li></ol>

The authors declare no conflict of interest.

At the scale of aquatic microorganisms, the environment is far from homogenous and is often characterized by physical/chemical gradients that structure microbial communities1,15. The capacity of motile microorganisms to use behavior (i.e., chemotaxis) facilitates foraging within these heterogeneous microenvironments1. Studying chemotaxis directly in the environment has the potential to identify important interspecific interactions and chem...

Diverse microorganisms use motility and chemotaxis to exploit patchy nutrient environments, find hosts, or avoid deleterious conditions1,2,3. These microbial behaviours can in turn influence rates of chemical transformation4 and promote symbiotic partnerships across terrestrial, freshwater, and marine ecosystems2,5.
Chemotaxis has been extensively studied under laboratory conditions for the past 60 years6. The first quantitative method to study chemotaxis, the capillary assay, involves a capillary tube filled with a putative chemoattractant immersed in a suspension of bacteria6. Diffusion of the chemical out of the tube creates a chemical gradient, and chemotactic bacteria respond to this gradient by migrating into the tube7. Since the development of the capillary assay, still widely used today, many other techniques have been developed to study chemotaxis under increasingly controlled physical/chemical conditions, with the most recent involving the use of microfluidics8,9,10.
Microfluidics, together with high-speed video microscopy, enables tracking of the behavior of single cells in response to carefully controlled gradients. Although these techniques have vastly improved our understanding of chemotaxis, they have been restricted to laboratory use and do not translate easily to field deployment in environmental systems. As a consequence, the capacity of natural communities of bacteria to use chemotaxis within natural ecosystems has not been examined; thus, current understanding of the potential ecological importance of chemotaxis is biased toward artificial laboratory conditions and a limited number of laboratory-cultured bacterial isolates. The recently developed ISCA overcomes these limitations11.
The ISCA builds on the general principle of the capillary assay; however, it makes use of modern microfabrication techniques to deliver a highly replicated, easily deployable experimental platform for the quantification of chemotaxis toward compounds of interest in the natural environment. It also allows identification and characterization of chemotactic microorganisms by direct isolation or molecular techniques. While the first working device was self-fabricated and constructed of glass and PDMS11, the latest injection-molded version is composed of polycarbonate, using a highly standardized fabrication procedure (for interest in the latest version of the device, the corresponding authors can be contacted).
The ISCA is credit card-sized and consists of 20 wells distributed in a 5 x 4 well array, each linked to the external aquatic environment by a small port (800 &#181;m in diameter; Figure 1). Putative chemoattractants loaded into the wells diffuse into the environment via the port, and chemotactic microbes respond by swimming through the port into the well. As many factors can influence the outcome of an ISCA experiment in the natural environment, this step-by-step protocol will help new users overcome potential hurdles and facilitate effective deployments.

<table>
	<tbody>
		<tr>
			<td>Acrylic glue</td>
			<td>Evonik</td>
			<td>1133</td>
			<td>Acrifix 1S 0116</td>
		</tr>
		<tr>
			<td>Acrylic sheet</td>
			<td>McMaster-Carr</td>
			<td>8505K725</td>
			<td>Or different company</td>
		</tr>
		<tr>
			<td>Adhesive tape</td>
			<td>Scotch</td>
			<td>3M 810</td>
			<td>Scotch Magic tape</td>
		</tr>
		<tr>
			<td>Autoclave</td>
			<td>Systec</td>
			<td>D-200</td>
			<td>Or different company</td>
		</tr>
		<tr>
			<td>Benchtop centrifuge</td>
			<td>Fisher Scientific</td>
			<td>75002451</td>
			<td>Or different company</td>
		</tr>
		<tr>
			<td>Bungee cord</td>
			<td>Paracord Planet</td>
			<td>667569184000</td>
			<td>Or different company</td>
		</tr>
		<tr>
			<td>Centrifuge tube - 2 mL</td>
			<td>Sigma Aldrich</td>
			<td>BR780546-500EA</td>
			<td>Eppendorf tube</td>
		</tr>
		<tr>
			<td>Conical centrifuge tube - 15 mL</td>
			<td>Fisher Scientific</td>
			<td>11507411</td>
			<td>Falcon tube</td>
		</tr>
		<tr>
			<td>Conical centrifuge tube - 50 mL</td>
			<td>Fisher Scientific</td>
			<td>10788561</td>
			<td>Falcon tube</td>
		</tr>
		<tr>
			<td>Deployment arm</td>
			<td>Irwin</td>
			<td>1964719</td>
			<td>Or different company</td>
		</tr>
		<tr>
			<td>Deployment enclosure plug</td>
			<td>Fisher Scientific</td>
			<td>21-236-4</td>
			<td>See alternatives in manuscript</td>
		</tr>
		<tr>
			<td>Disposable wipers</td>
			<td>Kimtech - Fisher Scientific</td>
			<td>06-666</td>
			<td>Kimwipes</td>
		</tr>
		<tr>
			<td>Flow cytometer</td>
			<td>Beckman</td>
			<td>C09756</td>
			<td>CYTOFlex</td>
		</tr>
		<tr>
			<td>Glutaraldehyde 25%</td>
			<td>Sigma Aldrich</td>
			<td>G5882</td>
			<td>Or different company</td>
		</tr>
		<tr>
			<td>Green fluorescent dye</td>
			<td>Sigma Aldrich</td>
			<td>S9430</td>
			<td>SYBR Green I - 1:10,000 final dilution</td>
		</tr>
		<tr>
			<td>Hydrophilic GP filter cartridge - 0.2 &#181;m</td>
			<td>Merck</td>
			<td>C3235</td>
			<td>Sterivex filter</td>
		</tr>
		<tr>
			<td>In Situ Chemotaxis Assay (ISCA)</td>
			<td>-</td>
			<td>-</td>
			<td>Contact corresponding authors</td>
		</tr>
		<tr>
			<td>Laser cutter</td>
			<td>Epilog Laser</td>
			<td>Fusion pro 32</td>
			<td>Or different company</td>
		</tr>
		<tr>
			<td>Luria Bertani Broth</td>
			<td>Sigma Aldrich</td>
			<td>L3022</td>
			<td>Or different company</td>
		</tr>
		<tr>
			<td>Marine Broth 2216</td>
			<td>VWR</td>
			<td>90004-006</td>
			<td>Difco</td>
		</tr>
		<tr>
			<td>Nylon slotted flat head screws</td>
			<td>McMaster-Carr</td>
			<td>92929A243</td>
			<td>M 2 &#215; 4 &#215; 8 mm</td>
		</tr>
		<tr>
			<td>Pipette set</td>
			<td>Fisher Scientific</td>
			<td>05-403-151</td>
			<td>Or different company</td>
		</tr>
		<tr>
			<td>Pipette tips - 1 mL</td>
			<td>Fisher Scientific</td>
			<td>21-236-2A</td>
			<td>Or different company</td>
		</tr>
		<tr>
			<td>Pipette tips - 20 &#181;L</td>
			<td>Fisher Scientific</td>
			<td>21-236-4</td>
			<td>Or different company</td>
		</tr>
		<tr>
			<td>Pipette tips - 200 &#181;L</td>
			<td>Fisher Scientific</td>
			<td>21-236-1</td>
			<td>Or different company</td>
		</tr>
		<tr>
			<td>Sea salt</td>
			<td>Sigma Aldrich</td>
			<td>S9883</td>
			<td>For artificial seawater</td>
		</tr>
		<tr>
			<td>Serological pipette - 50 mL</td>
			<td>Sigma Aldrich</td>
			<td>SIAL1490-100EA</td>
			<td>Or different company</td>
		</tr>
		<tr>
			<td>Syringe filter - 0.02 &#181;m</td>
			<td>Whatman</td>
			<td>WHA68091002</td>
			<td>Anatop filter</td>
		</tr>
		<tr>
			<td>Syringe filter - 0.2 &#181;m</td>
			<td>Fisher Scientific</td>
			<td>10695211</td>
			<td>Or different company</td>
		</tr>
		<tr>
			<td>Syringe needle 27G</td>
			<td>Henke Sass Wolf</td>
			<td>4710004020</td>
			<td>0.4 &#215; 12 mm</td>
		</tr>
		<tr>
			<td>Syringes - 1 mL</td>
			<td>Codau</td>
			<td>329650</td>
			<td>Insulin Luer U-100</td>
		</tr>
		<tr>
			<td>Syringes - 10 mL</td>
			<td>BD</td>
			<td>303134</td>
			<td>Or different company</td>
		</tr>
		<tr>
			<td>Syringes - 50 mL</td>
			<td>BD</td>
			<td>15899152</td>
			<td>Or different company</td>
		</tr>
		<tr>
			<td>Tube rack - 15 mL</td>
			<td>Thomas Scientific</td>
			<td>1159V80</td>
			<td>Or different company</td>
		</tr>
		<tr>
			<td>Tube rack - 50 mL</td>
			<td>Thomas Scientific</td>
			<td>1159V80</td>
			<td>Or different company</td>
		</tr>
		<tr>
			<td>Uncoated High-Speed Steel General Purpose Tap</td>
			<td>McMaster-Carr</td>
			<td>8305A77</td>
			<td>Or different company</td>
		</tr>
		<tr>
			<td>Vacuum filter - 0.2 &#181;m</td>
			<td>Merck</td>
			<td>SCGPS05RE</td>
			<td>Steritop filter</td>
		</tr>
	</tbody>
</table>

in situ chemotaxis assay to examine microbial behavior in aquatic ecosystems

Microbial behaviors, such as motility and chemotaxis (the ability of a cell to alter its movement in response to a chemical gradient), are widespread across the bacterial and archaeal domains. Chemotaxis can result in substantial resource acquisition advantages in heterogeneous environments. It also plays a crucial role in symbiotic interactions, disease, and global processes, such as biogeochemical cycling. However, current techniques restrict chemotaxis research to the laboratory and are not easily applicable in the field. Presented here is a step-by-step protocol for the deployment of the in situ chemotaxis assay (ISCA), a device that enables robust interrogation of microbial chemotaxis directly in the natural environment. The ISCA is a microfluidic device consisting of a 20 well array, in which chemicals of interest can be loaded. Once deployed in aqueous environments, chemicals diffuse out of the wells, creating concentration gradients that microbes sense and respond to by swimming into the wells via chemotaxis. The well contents can then be sampled and used to (1) quantify strength of the chemotactic responses to specific compounds through flow cytometry, (2) isolate and culture responsive microorganisms, and (3) characterize the identity and genomic potential of the responding populations through molecular techniques. The ISCA is a flexible platform that can be deployed in any system with an aqueous phase, including marine, freshwater, and soil environments.

This section presents laboratory results using the ISCA to test the chemotactic response of marine microbes to a concentration range of glutamine, an amino acid known to attract soil bacteria14. The concentration of glutamine that elicited the strongest chemotactic response in the laboratory tests was used to perform a chemotaxis assay in the marine environment.
To perform the laboratory tests, seawater communities sampled from coastal water in Sydney, Australia, were e...

Watch this Scientific Journal Video about In Situ Chemotaxis Assay to Examine Microbial Behavior in Aquatic Ecosystems at JoVE.com

In Situ Chemotaxis Assay to Examine Microbial Behavior in Aquatic Ecosystems

Presented here is the protocol for an in situ chemotaxis assay, a recently developed microfluidic device that enables studies of microbial behavior directly in the environment.

Microbial behaviors, such as motility and chemotaxis (the ability of a cell to alter its movement in response to a chemical gradient), are widespread ...

in-situ-chemotaxis-assay-to-examine-microbial-behavior-aquatic

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JoVE Journal

Environment

6.9K Views.  ETH Zürich. Presented here is the protocol for an in situ chemotaxis assay, a recently developed microfluidic device that enables studies of microbial behavior directly in the environment.

Video: In Situ Chemotaxis Assay to Examine Microbial Behavior in Aquatic Ecosystems

Optimize Flue Gas Settings to Promote Microalgae Growth in Photobioreactors via Computer Simulations

Flue gas from power plants can promote algal cultivation and reduce greenhouse gas emissions1. Microalgae not only capture solar energy more efficiently than plants3, but also synthesize advanced biofuels2-4. Generally, atmospheric CO2 is not a sufficient source for supporting maximal algal growth5. On the other hand, the high concentrations of CO2 in industrial exhaust gases have adverse effects on algal physiology. Consequently, both cultivation conditions (such as nutrients and light) and the control of the flue gas flow into the photo-bioreactors are important to develop an efficient &ldquo;flue gas to algae&rdquo; system. Researchers have proposed different photobioreactor configurations4,6 and cultivation strategies7,8 with flue gas. Here, we present a protocol that demonstrates how to use models to predict the microalgal growth in response to flue gas settings. We perform both experimental illustration and model simulations to determine the favorable conditions for algal growth with flue gas. We develop a Monod-based model coupled with mass transfer and light intensity equations to simulate the microalgal growth in a homogenous photo-bioreactor. The model simulation compares algal growth and flue gas consumptions under different flue-gas settings. The model illustrates: 1) how algal growth is influenced by different volumetric mass transfer coefficients of CO2; 2) how we can find optimal CO2 concentration for algal growth via the dynamic optimization approach (DOA); 3) how we can design a rectangular on-off flue gas pulse to promote algal biomass growth and to reduce the usage of flue gas. On the experimental side, we present a protocol for growing Chlorella under the flue gas (generated by natural gas combustion). The experimental results qualitatively validate the model predictions that the high frequency flue gas pulses can significantly improve algal cultivation.

Flue gas from power plants is a cheap CO2 source for algal growth. We have built prototype &#34;flue gas to algal cultivation&#34; systems and described how to scale up the algal cultivation process. We have demonstrated the use of a mass-transfer bio-reaction model to simulate and to design the optimal operation of flue gas for the growth of Chlorella sp. in algal photo-bioreactors.

Flue gas from power plants can promote algal cultivation and reduce greenhouse gas emissions1. Microalgae not only capture solar energy more efficiently ...

An Aquatic Microbial Metaproteomics Workflow: From Cells to Tryptic Peptides Suitable for Tandem Mass Spectrometry-based Analysis

Meta-omic technologies such as metagenomics, metatranscriptomics and metaproteomics can aid in the understanding of microbial community structure and metabolism. Although powerful, metagenomics alone can only elucidate functional potential. On the other hand, metaproteomics enables the description of the expressed in situ metabolism and function of a community. Here we describe a protocol for cell lysis, protein and DNA isolation, as well as peptide digestion and extraction from marine microbial cells collected on a cartridge filter unit (such as the Sterivex filter unit) and preserved in an RNA stabilization solution (like RNAlater). In mass spectrometry-based proteomics studies, the identification of peptides and proteins is performed by comparing peptide tandem mass spectra to a database of translated nucleotide sequences. Including the metagenome of a sample in the search database increases the number of peptides and proteins that can be identified from the mass spectra. Hence, in this protocol DNA is isolated from the same filter, which can be used subsequently for metagenomic analysis.

This protocol is for the extraction and concentration of protein and DNA from microbial biomass collected from seawater, followed by the generation of tryptic peptides suitable for tandem mass spectrometry-based proteomic analysis.

Meta-omic technologies such as metagenomics, metatranscriptomics and metaproteomics can aid in the understanding of microbial community structure and ...

Characterization and Application of Passive Samplers for Monitoring of Pesticides in Water

Five different water passive samplers were calibrated under laboratory conditions for measurement of 124 legacy and current used pesticides. This study provides a protocol for the passive sampler preparation, calibration, extraction method and instrumental analysis. Sampling rates (RS) and passive sampler-water partition coefficients (KPW) were calculated for silicone rubber, polar organic chemical integrative sampler POCIS-A, POCIS-B, SDB-RPS and C18 disk. The uptake of the selected compounds depended on their physicochemical properties, i.e., silicone rubber showed a better uptake for more hydrophobic compounds (log octanol-water partition coefficient (KOW) &#62; 5.3), whereas POCIS-A, POCIS-B and SDB-RPS disk were more suitable for hydrophilic compounds (log KOW &#60; 0.70).

A protocol about the characterization and application of five different passive sampling devices is presented.

Five different water passive samplers were calibrated under laboratory conditions for measurement of 124 legacy and current used pesticides. This study ...

Understanding Dissolved Organic Matter Biogeochemistry Through In Situ Nutrient Manipulations in Stream Ecosystems

Dissolved organic matter (DOM) is a highly diverse mixture of molecules providing one of the largest sources of energy and nutrients to stream ecosystems. Yet the in situ study of DOM is difficult as the molecular complexity of the DOM pool cannot be easily reproduced for experimental purposes. Nutrient additions to streams however, have been shown to repeatedly alter the in situ and ambient DOM pool. Here we demonstrate an easily replicable field-based method for manipulating the ambient pool of DOM at the ecosystem scale. During nutrient pulse experiments changes in the concentration of both dissolved organic carbon and dissolved organic nitrogen can be examined across a wide-range of nutrient concentrations. This method allows researchers to examine the controls on the DOM pool and make inferences regarding the role and function that certain fractions of the DOM pool play within ecosystems. We advocate the use of this method as a technique to help develop a deeper understanding of DOM biogeochemistry and how it interacts with nutrients. With further development this method may help elucidate the dynamics of DOM in other ecosystems.

Understanding Dissolved Organic Matter Biogeochemistry Through In Situ Nutrient Manipulations in Stream Ecosystems

Dissolved organic matter provides an important source of energy and nutrients to stream ecosystems. Here we demonstrate a field-based method to manipulate the ambient pool of dissolved organic matter in situ through easily replicable nutrient pulses.

Dissolved organic matter (DOM) is a highly diverse mixture of molecules providing one of the largest sources of energy and nutrients to stream ecosystems. ...

Double-stranded RNA Oral Delivery Methods to Induce RNA Interference in Phloem and Plant-sap-feeding Hemipteran Insects

Phloem and plant sap feeding insects invade the integrity of crops and fruits to retrieve nutrients, in the process damaging food crops. Hemipteran insects account for a number of economically substantial pests of plants that cause damage to crops by feeding on phloem sap. The brown marmorated stink bug (BMSB), Halyomorpha halys (Heteroptera: Pentatomidae) and the Asian citrus psyllid (ACP), Diaphorina citri Kuwayama (Hemiptera: Liviidae) are hemipteran insect pests introduced in North America, where they are an invasive agricultural pest of high-value specialty, row, and staple crops and citrus fruits, as well as a nuisance pest when they aggregate indoors. Insecticide resistance in many species has led to the development of alternate methods of pest management strategies. Double-stranded RNA (dsRNA)-mediated RNA interference (RNAi) is a gene silencing mechanism for functional genomic studies that has potential applications as a tool for the management of insect pests. Exogenously synthesized dsRNA or small interfering RNA (siRNA) can trigger highly efficient gene silencing through the degradation of endogenous RNA, which is homologous to that presented. Effective and environmental use of RNAi as molecular biopesticides for biocontrol of hemipteran insects requires the in vivo delivery of dsRNAs through feeding. Here we demonstrate methods for delivery of dsRNA to insects: loading of dsRNA into green beans by immersion, and absorbing of gene-specific dsRNA with oral delivery through ingestion. We have also outlined non-transgenic plant delivery approaches using foliar sprays, root drench, trunk injections as well as clay granules, all of which may be essential for sustained release of dsRNA. Efficient delivery by orally ingested dsRNA was confirmed as an effective dosage to induce a significant decrease in expression of targeted genes, such as juvenile hormone acid O-methyltransferase (JHAMT) and vitellogenin (Vg). These innovative methods represent strategies for delivery of dsRNA to use in crop protection and overcome environmental challenges for pest management.

This article demonstrates novel techniques developed for oral delivery of double-stranded RNA (dsRNA) through the vascular tissues of plants for RNA interference (RNAi) in phloem sap feeding insects.

Phloem and plant sap feeding insects invade the integrity of crops and fruits to retrieve nutrients, in the process damaging food crops. Hemipteran ...

Experimental Protocol for Examining Behavioral Response Profiles in Larval Fish: Application to the Neuro-stimulant Caffeine

Fish models and behaviors are increasingly used in the biomedical sciences; however, fish have long been the subject of ecological, physiological and toxicological studies. Using automated digital tracking platforms, recent efforts in neuropharmacology are leveraging larval fish locomotor behaviors to identify potential therapeutic targets for novel small molecules. Similar to these efforts, research in the environmental sciences and comparative pharmacology and toxicology is examining various behaviors of fish models as diagnostic tools in tiered evaluation of contaminants and real-time monitoring of surface waters for contaminant threats. Whereas the zebrafish is a popular larval fish model in the biomedical sciences, the fathead minnow is a common larval fish model in ecotoxicology. Unfortunately, fathead minnow larvae have received considerably less attention in behavioral studies. Here, we develop and demonstrate a behavioral profile protocol using caffeine as a model neurostimulant. Though photomotor responses of fathead minnows were occasionally affected by caffeine, zebrafish&#160;were markedly more sensitive for photomotor and locomotor endpoints, which responded at environmentally relevant levels. Future studies are needed to understand comparative behavioral sensitivity differences among fish with age and time of day, and to determine whether similar behavioral effects would occur in nature and be indicative of adverse outcomes at the individual or population levels of biological organization.

Here, we present a protocol to examine larval zebrafish and fathead minnow locomotor activities and photomotor responses (PMR) using an automated tracking software. When incorporated in common toxicology bioassays, analyses of these behaviors provide a diagnostic tool to examine chemical bioactivity. This protocol is described using caffeine, a model neurostimulant.

Fish models and behaviors are increasingly used in the biomedical sciences; however, fish have long been the subject of ecological, physiological and ...

Fluorescently Labeled Bacteria as a Tracer to Reveal Novel Pathways of Organic Carbon Flow in Aquatic Ecosystems

Elucidating trophic interactions, such as predation and its effects, is a frequent task for many researchers in ecology. The study of microbial communities has many limitations, and determining a predator, prey, and predatory rates is often difficult. Presented here is an optimized method based on the addition of fluorescently labelled prey as a tracer, which allows for reliable quantitation of the grazing rates in aquatic predatory eukaryotes and estimation of nutrient transfer to higher trophic levels.

Presented here is a protocol for a single-cell, epifluorescence microscopy-based technique to quantify grazing rates in aquatic predatory eukaryotes with high precision and taxonomic resolution.

Elucidating trophic interactions, such as predation and its effects, is a frequent task for many researchers in ecology. The study of microbial ...

Modeling the Size Spectrum for Macroinvertebrates and Fishes in Stream Ecosystems

The size spectrum is an inverse, allometric scaling relationship between average body mass (M) and the density (D) of individuals within an ecological community or food web. Importantly, the size spectrum assumes that individual size, rather than species&#8217; behavioral or life history characteristics, is the primary determinant of abundance within an ecosystem. Thus, unlike traditional allometric relationships that focus on species-level data (e.g., mean species&#8217; body size vs. population density), size spectra analyses are &#8216;ataxic&#8217; &#8211; individual specimens are identified only by their size, without consideration of taxonomic identity. Size spectra models are efficient representations of traditional, complex food webs and can be used in descriptive as well as predictive contexts (e.g., predicting responses of large consumers to changes in basal resources). Empirical studies from diverse aquatic ecosystems have also reported moderate to high levels of similarity in size spectra slopes, suggesting that common processes may regulate the abundances of small and large organisms in very different settings. This is a protocol to model the community-level size spectrum in wadable streams. The protocol consists of three main steps. First, collect quantitative benthic fish and invertebrate samples that can be used to estimate local densities. Second, standardize the fish and invertebrate data by converting all individuals to ataxic units (i.e., individuals identified by size, irrespective of taxonomic identity), and summing individuals within log2 size bins. Third, use linear regression to model the relationship between ataxic M and D estimates. Detailed instructions are provided herein to complete each of these steps, including custom software to facilitate D estimation and size spectra modeling.

This is a protocol to model the size spectrum (scaling relationship between individual mass and population density) for combined fish and invertebrate data from wadable streams and rivers. Methods include: field techniques to collect quantitative fish and invertebrate samples; lab methods to standardize the field data; and statistical data analysis.

The size spectrum is an inverse, allometric scaling relationship between average body mass (M) and the density (D) of individuals within an ecological ...

RNA Interference in Aquatic Beetles as a Powerful Tool for Manipulating Gene Expression at Specific Developmental Time Points

RNA interference (RNAi) remains a powerful technique that allows for the targeted reduction of gene expression through mRNA degradation. This technique is applicable to a wide variety of organisms and is highly efficient in the species-rich order Coleoptera (beetles). Here, we summarize the necessary steps for developing this technique in a novel organism and illustrate its application to the different developmental stages of the aquatic diving beetle Thermonectus marmoratus. Target gene sequences can be obtained cost-effectively through the assembly of transcriptomes against a close relative with known genomics or de novo. Candidate gene cloning utilizes a specific cloning vector (the pCR4-TOPO plasmid), which allows the synthesis of double-stranded RNA (dsRNA) for any gene with the use of a single common primer. The synthesized dsRNA can be injected into either embryos for early developmental processes or larvae for later developmental processes. We then illustrate how RNAi can be injected into aquatic larvae using immobilization in agarose. To demonstrate the technique, we provide several examples of RNAi experiments, generating specific knockdowns with predicted phenotypes. Specifically, RNAi for the tanning gene laccase2 leads to cuticle lightening in both larvae and adults, and RNAi for the eye pigmentation gene white produces a lightening/lack of pigmentation in eye tubes. In addition, the knockdown of a key lens protein leads to larvae with optical deficiencies and a reduced ability to hunt prey. Combined, these results exemplify the power of RNAi as a tool for investigating both morphological patterning and behavioral traits in organisms with only transcriptomic databases.

RNA interference is a widely applicable, powerful technique for manipulating gene expression at specific developmental stages. Here, we describe the necessary steps for implementing this technique in the aquatic diving beetle Thermonectus marmoratus, from the acquisition of gene sequences to the knockdown of genes that affect structure or behavior.

RNA interference (RNAi) remains a powerful technique that allows for the targeted reduction of gene expression through mRNA degradation. This technique is ...

Simulating Impacts of Ice Storms on Forest Ecosystems

Ice storms can have profound and lasting effects on the structure and function of forest ecosystems in regions that experience freezing conditions. Current models suggest that the frequency and intensity of ice storms could increase over the coming decades in response to changes in climate, heightening interest in understanding their impacts. Because of the stochastic nature of ice storms and difficulties in predicting when and where they will occur, most past investigations of the ecological effects of ice storms have been based on case studies following major storms. Since intense ice storms are exceedingly rare events it is impractical to study them by waiting for their natural occurrence. Here we present a novel alternative experimental approach, involving the simulation of glaze ice events on forest plots under field conditions. With this method, water is pumped from a stream or lake and sprayed above the forest canopy when air temperatures are below freezing. The water rains down and freezes upon contact with cold surfaces. As the ice accumulates on trees, the boles and branches bend and break; damage that can be quantified through comparisons with untreated reference stands. The experimental approach described is advantageous because it enables control over the timing and amount of ice applied. Creating ice storms of different frequency and intensity makes it possible to identify critical ecological thresholds necessary for predicting and preparing for ice storm impacts.

Ice storms are important weather events that are challenging to study because of difficulties in predicting their occurrence. Here, we describe a novel method for simulating ice storms that involves spraying water over a forest canopy during sub-freezing conditions.

Ice storms can have profound and lasting effects on the structure and function of forest ecosystems in regions that experience freezing conditions. ...