Name: macro-rheology characterization of gill raker mucus in the silver carp, hypophthalmichthys molitrix
Uploaded: 2020-07-10T14:00:00.000+00:00
Duration: 9 min 13 s
Description: Watch this Scientific Journal Video about Macro-Rheology Characterization of Gill Raker Mucus in the Silver Carp, Hypophthalmichthys molitrix at JoVE.com

The authors acknowledge support and funding from the GW Center for Biomimetics and Bioinspired Engineering. We thank Professor L. Patricia Hernandez of the Department of Biological Sciences at The George Washington University for inspiring the investigation and ongoing collaboration, providing biological expertise on the physiology of the silver carp and providing the mucus samples. We thank the students, Mr. David Palumbo, Ms. Carly Cohen, Mr. Isaac Finberg, Mr. Dominick Petrosino, Mr. Alexis Renderos, Ms. Priscilla Varghese, Mr. Carter Tegen and Mr. Raghav Pajjur for help in the laboratory and Mr. Thomas Evans and Mr. James Thomas of TA Instruments, New Castle, DE for support with training and maintenance of the rheometer. Images for Figures 5A,C were taken during a dissection performed by Professor L. Patricia Hernandez of the Department of Biological Sciences at The George Washington University.

1. Preparation of the mucus solutions of various concentrations
NOTE: Three concentrations of the mucus solution (400 mg/mL, 200 mg/mL and 100 mg/mL with approximate volumes, 1 mL, 1 mL, and 2 mL, respectively) are prepared for this experiment.&#160;
<ol>
	<li>To calculate the mass of the mucus, measure the average mass of the vials with (Mwith-mucus ; mg) and without mucus (Mvials ; mg). Then subtract the mass of the vials with mucus with that without mucus (Mmucus = Mwith-mucus - Mvials ; mg).&#160;&#160;</li>
	<li>Dilute the mucus into three concentrations (400, 200, 100, mg/mL) with deionized (DI) water.
	<ol>
		<li>Prepare the first concentration of the mucus solution, 400 mg/mL by adding 0.6 mL DI water to the mucus using a micropipette. 
		NOTE: Since the approximate volume of the extracted mucus was 1.4 mL, the 400 mg/mL solution will have a total volume of ~ 2 mL.</li>
		<li>Place the 400 mg/mL mucus solution vial on a shaker to make sure that the mucus solution is adequately homogenized, and any mucus particulate agglomeration is mitigated.</li>
		<li>Prepare the second concentration of the mucus solution, 200 mg/mL, by drawing half the volume of the first-concentration mucus solution into a new vial using a micropipette and adding 1 mL of DI water into the new vial.</li>
		<li>Repeat step 1.2.2 for the first and second vials with mucus solutions.</li>
		<li>Prepare the third concentration of the mucus solution, 100 mg/mL, by drawing half the volume (1 mL) of the 200 mg/mL solution into a new vial using a micropipette and add of DI water into the new vial.</li>
		<li>Repeat step 1.2.2 for all three concentrations of mucus solutions in their respective vials (see Supplementary Figure 1).</li>
		<li>Store the mucus solution vials in a refrigerator until the rheometer calibration and testing is performed.</li>
	</ol>
	</li>
</ol>
2. Measurements and data acquisition using a rheometer
NOTE: The software used in this protocol for instrument control and data acquisition with rheometer are noted in the Table of Materials. This software will be referred as &#8216;rheometer instrument control software&#8217;.
<ol>
	<li>Set up and calibrate the rheometer instrument.
	<ol>
		<li>Turn on the compressed air supply to the rheometer and make sure the pneumatic table and the rheometer are leveled using a bubble gauge. Twist off the protective cap on the rheometer shaft and hold shaft still whilst unscrewing.</li>
		<li>Turn on the rheometer main switches to activate the magnetic bearings on the rheometer.</li>
		<li>Turn on the rheometer control computer with the rheometer instrument control software installed in it and launch the rheometer instrument control software (see Supplementary Figure 2).</li>
		<li>Perform instrument calibration by selecting the tabs, &#39;Calibration | Instrument&#39; from the software window. Choose &#39;Instrument&#39; option. Click on &#39;Calibrate&#39; under &#8216;Inertia&#8217;. Record the instrument inertia calibration value in &#956;N.m.s2 and repeat calibration at least 3x to ensure calibration values are within 10% of each other (see Supplementary &#160;Figure 3).</li>
	</ol>
	</li>
	<li>Install the rheometer geometry on the shaft of the rheometer.
	<ol>
		<li>Click the &#8216;Geometries&#8217; tab in the rheometer instrument control software.</li>
		<li>Clean the cone with the desired geometry, (40 mm diameter, 1&#9702; 0&#8217; 11&#8217;&#8217; cone) and Peltier plate with isopropanol (see Table 1, Table of Materials, Figure 1, and Figure 2). 
		NOTE: The Peltier Plate comes installed on the rheometer; it can be cleaned with isopropanol while it is directly fixed to the rheometer.</li>
		<li>Ensure that the Peltier plate fixture is free from any visible dust and clean, if necessary, with isopropanol. Install the Peltier plate if it is not pre-installed in the rheometer and connect the heat sink connections.</li>
		<li>Press the &#8216;Lock button&#8217; on rheometer to the lock shaft that is connected to the cone geometry. This arrests the position of the shaft, but it can rotate freely at the position.</li>
		<li>Click on &#8216;Smart Swap | Enabled&#8217; in the rheometer instrument control software tab to allow automatic detection of the geometry (see Supplementary Figure 4).</li>
		<li>Turn the shaft on top of the rheometer to screw on geometry. The software will detect the 40 mm diameter, 1&#9702; 0&#8217; 11&#8217;&#8217; cone angle geometry at this stage (see Table 1 and Table of Materials).</li>
		<li>Repeat steps 2.2.5 &#8211; 2.2.6 to ensure that the geometry is detected.</li>
		<li>Select &#8216;Gap&#8217; under the &#8216;Control Panel&#8217; of the rheometer instrument control software, click on &#8216;Options&#8217; icon and chose &#8216;Axial Force&#8217; option. Set axial force to &#8216;1 Newton&#8217;; this is to ensure the cone geometry touches the Peltier plate for zero gap initialization (see Supplementary Figure 5).</li>
	</ol>
	</li>
	<li>Perform the rheometer geometry calibration.
	<ol>
		<li>Select the tab, &#39;Geometry&#39; from the software window. Click on &#39;Calibrate&#39; under &#8216;Inertia&#8217;. Record the geometry inertia calibration value in &#956;N.m.s2 and repeat this 2-3 times to ensure calibration values are within 10% of each other.</li>
		<li>Click on &#39;Calibrate&#39; under &#8216;Friction&#8217; in the software window. Record the geometry friction calibration value in &#956;N.m/(rad/s) and repeat this 2-3 times to ensure calibration values are within 10% of each other (see Supplementary Figure 6).</li>
	</ol>
	</li>
	<li>Perform the zero-gap initialization 
	NOTE: Since the geometry cannot be accurately raised above the Peltier Plate to perform measurements without a reference &#8220;zero&#8221; position, zero-gap initialization is performed. For the measurement purposes, the geometry has a built-in geometry gap of 24 &#181;m and a trim gap of 28 &#181;m. The trim gap is set to effectively clean the excess fluid that may spill outside the surface area of the geometry. These gaps are imperative for accurately measuring data using the sample and the rheometer. The step 2.4.1 is absolutely required to make sure that the geometry is set to zero gap for achieving the geometry and trim gaps of 24 &#181;m and 28 &#181;m, respectively.
	<ol>
		<li>Click on the &#8216;Zero gap&#8217; icon under &#8216;Gap&#8217; tab in the &#8216;Control Panel&#8217; in the software window. The initialization is complete when the axial force experienced by the geometry is greater than or equal to 1 N, as it touches the Peltier plate. Ensure that the rheometer gap is zeroed so that its reference position is accurate (see Supplementary Figure 7 and Supplementary Figure 8).</li>
		<li>Press &#8216;up and down arrow&#8217; controls on the rheometer instrument or &#8216;geometry raise and lower&#8217; icons under the &#8216;Gap&#8217; tab in the rheometer instrument control software to raise the geometry to any arbitrary height. The control screen on the rheometer instrument and the control panel of the rheometer instrument control software will display the (same) gap height.</li>
	</ol>
	</li>
	<li>Set up the experimental procedure in the rheometer instrument control software. Perform the characterization of rheological properties by using a cone-on-Peltier plate geometry at 22 &#176;C. 
	NOTE: The US Geological Survey website was used to ascertain the river water temperature River on September 20, 2018, when the silver carp used for the GR mucus experiments were fished at the Hart Creek location36. The temperature of the mucus can affect the rheological properties. The significance of adjusting the values to river temperature is to approximately match the temperature under which the mucus properties can be realistically estimated. &#160;
	<ol>
		<li>Select the tab, &#39;Experiments&#39; in the rheometer instrument control software and fill in the relevant information such as &#39;Name&#39;, &#39;Operator&#39;, &#39;Project&#39; etc. &#160;(see Supplementary Figure 9)</li>
		<li>Select the tab, &#39;Geometry&#39; and make sure the information agrees with steps 2.2.5. - 2.2.7. (see Supplementary Figure 10).</li>
		<li>Select the tab, &#39;Procedure&#39;, and use the arrow keys set up &#39;1: Oscillation Amplitude&#39; procedure. (see Supplementary Figure 11).</li>
		<li>Initialize &#39;Environmental Control&#39; settings as the following: &#39;Temperature = 22 &#176;C&#39;; &#39;Soak Time = 120 s&#39; and check the box &#39;Wait for Temperature&#39; (see Supplementary Figure 11).</li>
		<li>Initialize &#39;Test Parameters&#39; settings as the following: &#39;Frequency = 1 Hz&#39;; set &#39;Logarithmic sweep&#39;; &#39;Torque = 10 to 10000 &#956;N.m&#39;; &#39;Points per decade = 5&#39; (see Supplementary Figure 11).</li>
	</ol>
	</li>
	<li>Set up the experiment to determine the Linear Viscoelastic Range (LVR) of the mucus of known concentration (100 mg/mL)
	<ol>
		<li>Using an appropriate micropipette and pipette tip draw approximately 0.3 mL of fish mucus solution of concentration 100 mg/mL (see Step 1.2, Table of Materials).</li>
		<li>Introduce the mucus solution on to the Peltier plate using the micropipette (see Figure 2).</li>
		<li>Press &#8216;Trim Gap&#8217; button on rheometer to lower the geometry on to the Peltier plate. Alternatively, click on &#8216;Trim Gap&#8217; icon under &#8216;Gap&#8217; tab in the &#8216;Control Panel&#8217; option in the rheometer instrument control software (see Supplementary Figure 12).</li>
		<li>Use the micropipette with the pipette tip to remove any excess mucus solution and ensure that the fluid is underneath the geometry without any spilling near the periphery of the geometry. 
		NOTE: Improper loading of the fluid will lead to errors in the measurements. Under filled sample will lower torque distribution and over filled sample will lead erroneous stress distributions due to spilling along the edges.</li>
		<li>Select &#8216;Motor&#8217; and &#8216;Velocity&#8217; tabs to 5 rad/s and 0 rad/s alternately, until there is minimal inertia and velocity in the sample underneath the geometry. The control screen on the rheometer instrument and the control panel of the rheometer instrument control software will display the velocity (see Supplementary Figure 13).</li>
		<li>Press &#8216;Geometry Gap&#8217; button on rheometer to lower geometry to the preset suitable gap per specific geometry. Alternatively, click on &#8216;Geometry Gap&#8217; icon under &#8216;Gap&#8217; tab in the &#8216;Control Panel&#8217; option in the rheometer instrument control software (see Supplementary Figure 14).</li>
	</ol>
	</li>
	<li>Run the experiment to determine the Linear Viscoelastic Range (LVR) of the mucus of the known concentration (100 mg/mL).
	<ol>
		<li>Click &#8216;Start&#8217; icon on the rheometer instrument control software (see Supplementary Figure 15). 
		NOTE: The rheometer performs automatic measurements; once the &#8216;Start&#8217; button is pressed, the rheometer will take approximately 20 min to complete the test. The &#8216;Points per decade&#8217; setting in Step 2.5.5 determines how much time the rheometer will need to complete measurements.</li>
		<li>Run the experiment by clicking &#8216;Yes&#8217; on the pop-up box that appears and suggests that the geometry gap be lowered to the correct distance to start the experiment, if not already lowered. &#160;</li>
		<li>Observe the real time plot generated by the rheometer that reports the storage (G&#8217;) and loss (G&#8217;&#8217;) moduli. 
		NOTE: The G&#8217; and G&#8221; are the storage and loss moduli, respectively. The storage modulus represents the tendency for the material to recover its original shape following stress-induced deformation and is equivalent to elasticity. The loss modulus represents the extent to which the material resists the tendency to flow and is representative of fluid viscosity (see Figure 4).</li>
		<li>Set the X-axis of the plot to &#8216;Oscillation strain percentage&#8217;. To do this, right click on the graph presented and choose the &#8216;Graph Variables&#8217; tab (see Supplementary Figure 16).</li>
		<li>Record the oscillation strain percentage range from the plot before material enters the Non-Linear Viscoelastic range, once the test is complete.</li>
		<li>Press &#8216;up and down arrow&#8217; controls on the rheometer instrument or &#8216;geometry raise and lower&#8217; icons under the &#8216;Gap&#8217; tab in the rheometer instrument control software to raise the geometry to any arbitrary height above the Peltier plate.</li>
		<li>Save the file that contains both the experimental procedure and results in the native file format of the rheometer instrument control software to ascertain the linear viscoelastic region (LVR) of the mucus sample. 
		NOTE: This can be done by setting the X-axis of the plot to strain amplitude (%) and/or oscillation stress <img alt="Equation omega" src="/files/ftp_upload/61379/61379eqomega.jpg" style="font-size: 14px;" />&#160;before the data enters the non-linear viscoelastic region (NLVR) (see Supplementary Figure 16).</li>
	</ol>
	</li>
	<li>Run the dynamic sweeps and steady state shear rate flow test experiments in Linear Viscoelastic Range (LVR) for the mucus of known concentration 100, mg/mL to generate results from three independent mucus samples of 100 mg/mL. Perform these steps on the available mucus concentration samples individually.&#160;
	<ol>
		<li>Repeat steps 2.5.1 &#8211; 2.5.4.</li>
		<li>Initialize &#39;Test Parameters&#39; settings as the following: &#39;Frequency = 1 Hz&#39;; set &#39;Logarithmic sweep&#39;; &#39;Strain % = 100 to 10000 %; &#39;Points per decade = 10&#39;.</li>
		<li>Select the &#39;Procedure&#39; tab and use the arrow keys set up &#39;2: Oscillation Frequency&#39; procedure.</li>
		<li>Initialize &#39;Environmental Control&#39; settings as the following: &#39;Temperature = 22 &#176;C&#39;; &#39;Soak Time = 0.0 s&#39;.</li>
		<li>Initialize &#39;Test Parameters&#39; settings as the following: &#39;Strain % = 1 %&#39;; set &#39;Logarithmic sweep&#39;; &#39;Frequency = 20 to 1 Hz&#39;; &#39;Points per decade = 10&#39;.</li>
		<li>Select the &#39;Procedure&#39; tab and use the arrow keys set up &#39;3: Flow Sweep&#39; procedure.</li>
		<li>Initialize &#39;Environmental Control&#39; settings as the following: &#39;Temperature = 22 &#176;C&#39;; &#39;Soak Time = 0.0 s&#39;.</li>
		<li>Initialize &#39;Test Parameters&#39; settings as the following: &#39;Shear rate = 1 to 10000 1/s&#39;; &#39;Points per decade = 10&#39;; check box &#8216;Steady state sensing&#8217;.</li>
		<li>Repeat steps 2.7.1 &#8211; 2.7.2 and wait until the experiment is complete, approximately 45 minutes.</li>
		<li>Press &#8216;up and down arrow&#8217; controls on the rheometer instrument or &#8216;geometry raise and lower&#8217; icons under &#8216;Gap&#8217; tab in the rheometer instrument control software to raise the geometry to any arbitrary height.</li>
		<li>Use disposable wipes and gloves to remove and clean the mucus on the Peltier plate with isopropanol solution (see Table of Materials).</li>
		<li>Save the file that contains both the experimental procedure and results in the native file format of the rheometer instrument control software.&#160;</li>
	</ol>
	</li>
</ol>
3. Repeat the protocol for other concentrations of mucus solutions of 200 mg/mL and 400 mg/mL.
<ol>
	<li>Perform steps 2.5 &#8211; 2.8 including all the sub-steps listed therein for the remaining two concentrations of mucus solutions, 200 mg/mL and 400 mg/mL. &#160;</li>
</ol>
4. Graphical representation and data analysis
NOTE: The code provided in the supplemental code file performs data averaging and generates repeatability-errors, overlays the data from all experiments. The standard-deviation calculation features are not available in the rheometer instrument control software. The code is written in a programming language for data analysis, post-processing and graphical representation (see Table of Materials for details).&#160;&#160;&#160;
<ol>
	<li>Export data generated from step 2.8 pertaining to the 100 mg/mL GR mucus concentration and step 3.1 pertaining to the 200mg/mL and 400 mg/mL GR mucus concentrations &#160;into spreadsheet-format by clicking on the tab, &#8216;File | Export | Excel&#8217; in the rheometer instrument control software (see Supplementary Figure 17).</li>
	<li>Run supplemental codes to generate plots of apparent viscosity (&#951;) for varying shear strain rates (<img alt="Equation y" src="/files/ftp_upload/61379/61379eqy.jpg" />) and loss modulus (G&#8221;), storage modulus (G&#8217;) and phase angle (&#948;) for varying oscillation stress (<img alt="Equation omega" src="/files/ftp_upload/61379/61379eqomega.jpg" />) and generate representative results.</li>
</ol>

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J., Shephard, K. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Measurement+of+the+adhesion+of+a+desmid+to+a+substrate.">Measurement of the adhesion of a desmid to a substrate.</a> British Phycology Journal. 24, 293-295 (1989).</li><li>Sanderson, S. L., Cech, J. J., Patterson, M. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fluid+dynamics+in+suspension+feeding+black+fish.">Fluid dynamics in suspension feeding black fish.</a> Science. 251, 1346-1348 (1991).</li><li>Lai, S. K., Wang, Y. Y., Wirtz, D., Hanes, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Micro-+and+macrorheology+of+mucus.">Micro- and macrorheology of mucus.</a> Advanced Drug Delivery Reviews. 61 (2), 86-100 (2009).</li><li>Chaudhary, G., Ewoldt, R. H., Thiffeault, J. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Unravelling+hagfish+slime.">Unravelling hagfish slime.</a> Journal of Royal Society Interface. 16 (150), 20180710 (2019).</li><li>Downing, S., Salo, W., Spitzer, R., Koch, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+hagfish+slime+gland:+a+model+system+for+studying+the+biology+of+mucus.">The hagfish slime gland: a model system for studying the biology of mucus.</a> Science. 214, 1143-1145 (1981).</li><li>Hwang, S. H., Litt, M., Forsman, W. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rheological+properties+of+mucus.">Rheological properties of mucus.</a> Rheologica Acta. 8, 438-448 (1969).</li><li>Litt, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mucus+rheology.">Mucus rheology.</a> Archives of Internal Medicine. 126, 417-423 (1970).</li><li>Quarishi, M. S., Jones, N. S., Mason, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+rheology+of+nasal+mucus:+a+review.">The rheology of nasal mucus: a review.</a> Clinical Otolaryngology. 23, 403-413 (1998).</li><li>Nordg&#229;rd, C. T., Draget, K. I., Seternes, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rheology+of+salmon+skin+mucus.">Rheology of salmon skin mucus.</a> Annual Transactions - The Nordic Rheology Society. 23, 175-179 (2015).</li><li>Fern&#225;ndez-Alacid, L., 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=Comparison+between+properties+of+dorsal+and+ventral+skin+mucus+in+Senegalese+sole:+Response+to+an+acute+stress.">Comparison between properties of dorsal and ventral skin mucus in Senegalese sole: Response to an acute stress.</a> Aquaculture. 513, 734410 (2019).</li><li>Y&#252;ce, C., Willenbacher, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Challenges+in+Rheological+Characterization+of+Highly+Concentrated+Suspensions+-+Case+Study+for+Screen-printing+Silver+Pastes.">Challenges in Rheological Characterization of Highly Concentrated Suspensions - Case Study for Screen-printing Silver Pastes.</a> Journal of Visualized Experiments. (122), e55377 (2017).</li><li>Sultan, S., Mathew, A. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=3D+Printed+Porous+Cellulose+Nanocomposite+Hydrogel+Scaffolds.">3D Printed Porous Cellulose Nanocomposite Hydrogel Scaffolds.</a> J. Vis. Exp. (146), (2019).</li><li>Barnes, H. A., Hutton, J. F., Walters, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> An Introduction to Rheology. , (1989).</li><li>USGS Current Conditions for USGS 06910450 Missouri River at Jefferson City, MO. U.S. Geological Survey Available from: <a target="_blank" href="https://nwis.waterdata.usgs.gov/usa/nwis/uv/?cb_00010=on&amp;cb_00060=on&amp;cb_00065=on&amp;format=gif_default&amp;site_no=0691045&amp;p09-19&amp;end_date=2018-09-21">https://nwis.waterdata.usgs.gov/usa/nwis/uv/?cb_00010=on&amp;cb_00060=on&amp;cb_00065=on&amp;format=gif_default&amp;site_no=0691045&amp;p09-19&amp;end_date=2018-09-21</a> (2020)</li></ol>

No conflicts of interest declared.

One of the main objectives of developing this protocol is to establish that it is well-suited for rheological characterization of GR mucus when very small sample volumes are available. We acknowledge that more samples from a school of silver carp are needed to fully characterize the rheological properties of the GR mucus and the data presented herein are not a generalization across the entire silver carp population. Our technique is justified because of its efficacy with rheological characterization of small sample volumes and with extended investigations involving larger ensembles of mucus samples.
The critical steps within the protocol are the preparation of mucus solutions of various concentrations, measurements and data acquisition using a rotational rheometer, and graphical representation and data analysis for physical insights.
Physical insights into GR mucus data are drawn from graphical representations shown in Figure 3 and Figure 4, that are annotated with attributes of the expected material behavior. Zero-shear strain rate viscosity (&#951;o) values can be observed at low-shear strain rates where mobility of the material molecules dominates (Figure 3A and Figure 8A). Infinite-shear strain viscosity (&#951;&#8734;) values in non-Newtonian fluids are orders of magnitudes lower than the zero-shear strain rate viscosity. These data can be noticed at high shear rates where there is little or no dependence on intermolecular interactions (Figure 3A and Figure 8A). For non-Newtonian fluids, apparent viscosities progressively decrease as the shear rates increase and attain a constant low value (Figure 3A and Figure 8A). Yielding behavior in the GR mucus under steady state measurements&#160; can be represented with slope as shown in Figure 3A and presented in Equation 1., where &#951;a represents the apparent viscosity, &#963;y is the (constant) yield stress and <img alt="Equation y" src="/files/ftp_upload/61379/61379eqy.jpg" /> is the shear strain rate.
<img alt="Equation 1" src="/files/ftp_upload/61379/61379eq1.jpg" />
Figure 3A and Figure 8B are presented on a log-log scale and therefore, Equation 1 attains the following form:
<img alt="Equation 2" src="/files/ftp_upload/61379/61379eq2.jpg" />
where k &#8211; represents the apparent yield stress. On a log-log scale, the apparent viscosity decreases with a slope of &#8216;-1&#8217; indicates the material yield as shown in Figure 3A10.&#160; The 200 mg/mL and 400 mg/mL mucus concentrations possessed slopes of -1.8 and -0.91, respectively, and demonstrate the yielding behavior (Figure 8A). Under dynamic oscillation measurements, the viscoelastic characteristics are independent of the strain amplitude in the Linear Viscoelastic Region (LVR) (Figure 4). The yielding behavior in the GR mucus under dynamic oscillation measurements can be observed as the viscoelastic material (GR mucus) enters the non-linear viscoelastic region (NLVR) as the storage modulus (G&#8217;) decreases (Figure 4). In the NLVR regime the viscoelastic material will demonstrate solid-gel-like behavior if the storage modulus is greater than the loss modulus (G&#8217; &#62; G&#8221;). When the loss modulus exceeds the storage modulus (G&#8217; &#60; G&#8221;), a &#8220;crossover&#8221; between G&#8217; and G&#8221; data occur. As shown in Figure 7B,C, the 200 mg/mL and 400 mg/mL GR mucus concentrations demonstrated fluid-like behavior marked by the &#8220;crossover&#8221; between G&#8217; and G&#8221; data. The apparent yield stress under steady state measurements is represented as the average value of stress until an inflection point is reached (Figure 3B). Thereafter, the stress begins to increase sharply with an increase in the shear strain rate as shown in Figure 3B and Figure 8B. The GR mucus data (200 mg/mL and 400 mg/mL concentrations) showed shear-thinning fluid behavior until the material begins to yield (Figure 8A,B). The apparent yield stress was observed clearly in 200 mg/mL and 400 mg/mL mucus concentrations due to their non-Newtonian characteristics (Figure 8B). The apparent yield stress under dynamic oscillation measurements are shown in Figure 4 and Figure 7B,C as the &#8220;crossover&#8221; region between G&#8217; and G&#8221; data, followed by G&#8221; values exceeding G&#8217;. The 400 mg/mL GR mucus data showed shear-thinning, non-Newtonian behavior. The onset point of material yield was observed with an apparent yield stress of approximately 0.2736 Pa (Figure 7C). The hydrogel-to-fluid like transition with phase angle (&#948; = tan-1 (G&#8221;/G&#8217;)) changes are presented in Figures 4 and 7D-F. The extrema in the phase angle is associated with a Hookean solid at 0&#730; and viscous fluid at 90&#730; as shown in Figure 4. The phase angle values around 45&#730; were attributed to transition of gel-like behavior of the material to a fluid-like behavior. The 400 mg/mL mucus concentration clearly showed a change in the material characteristic from hydrogel to fluid like behavior through the process of yielding with an apparent yield stress of ~ 0.2736 Pa (Figure 7F).
Understanding the measurement limitations and avoiding data unsuitable for physical interpretation is a challenge with complex and soft biological fluids, especially in studies involving small sample volumes11. The data generated under low-torque and secondary flow effects are unsuitable for physical interpretation and are dependent on the geometry used in the rheometer (such as cone and plate in this study). These regimes were identified to avoid any misrepresentation of experimental data suffering from instrument resolution and measurement artifacts due to momentum diffusion. Low-torque limits (Figure 6A and Figure 8A) are functions of geometry and minimum torque generated by the instrument (Table 1). Under steady shear measurement conditions, the criterion for rejecting data affected by the low-torque limit for a cone-plate geometry of radius (R) with minimum torque (Tmin = 10 x 10-9 Nm, Table 1) has been discussed by Ewoldt et al. and is presented below11:
<img alt="Equation 3" src="/files/ftp_upload/61379/61379eq3.jpg" />
where <img alt="Equation y" src="/files/ftp_upload/61379/61379eqy.jpg" /> is the shear strain rate. &#160;Unlike the 100 mg/mL GR mucus concentrations, the 200 mg/ml and 400 mg/mL GR mucus concentrations were unaffected by low-torque effects clearly demonstrate non-Newtonian, shear thinning behavior with high zero-shear strain rate viscosities at low shear strain rates. The criterion for minimum measurable viscoelastic moduli under dynamic oscillation measurements has been discussed by Ewoldt et al. and presented below (Equation 4)11. In Equation 4, for a cone-plate geometry of radius (R) the minimum torque under oscillatory shear (Tmin = 2 x 10-9 Nm, Table 1).
<img alt="Equation 4" src="/files/ftp_upload/61379/61379eq4.jpg" />
where Gmin is the storage modulus (G&#8217;) or loss modulus (G&#8221;) and is the shear strain rate. The regimes of instrumentation limitation governed by low-torque effects are marked in Figure 6A and 6B. The secondary flow regime under steady state measurements is governed by an inward momentum diffusion of the fluid by means of an eddy residing within the rotational cone and plate geometry11. The secondary flow pattern increases torque incorrectly making the fluid appear to be shear-thickening (Figure 8A). The secondary flow limit, proposed by Ewoldt et al. in Figure 8A was drawn using the following relation11:
<img alt="Equation 5" src="/files/ftp_upload/61379/61379eq5.jpg" />
where L = &#946;R, &#946; is the cone angle, R is the cone radius, &#961; = 1000 kg m-3, Recrit = 4 and <img alt="Equation y" src="/files/ftp_upload/61379/61379eqy.jpg" /> is the shear rate. This regime helped in estimating the infinite-shear strain viscosity (&#951;&#8734;) values in GR mucus samples.
A modification of the protocol can be made by using a flat-plate geometry instead of the cone-plate geometry as shown in protocol presented herein. The flat-plate tests should be performed with a parametric variation of the measurement gap in the rotational rheometer to reveal the dependence of apparent yield stress on the measurement gap and geometry. The suggested improvements of the protocol presented in this paper are described below. A parametric variation of the strain amplitude in the linear viscoelastic regime (LVR) and oscillation frequency should be performed. &#8216;Tack and peel&#8217; rheology tests should be performed to develop a full understanding of the adhesivity of the GR mucus. Rheology characteristics of GR mucus should be performed on larger sample volume ensembles along with studies to measure any traces of blood cells to account its effect on the overall GR rheological properties.
The limitations of the protocol are described below. The intricacies of the GR mucus extraction-procedures and the presence of blood cells or tissue fragments in the mucus samples may influence the rheology of the mucus. However, it should be noted the mucus used in the protocol did not have any visible traces of blood. The GR mucus sample is a heterogeneous material and can possess different rheological properties due to the variance in location of and the conditions post-extraction. This limitation was addressed by sufficiently homogenizing the GR mucus using a shaker to breakdown any large clumps of mucus and tissue presence. Another important limitation is the very small GR mucus sample volumes (approximately 1.4 mL), harvested for analyses that constrain a generalization of GR mucus properties.
The significance of this protocol is that it allows for an accurate rheological characterization of non-Newtonian, biological fluids such as the mucus. The protocol presented herein paves the way for investigating other similar biological fluids associated with human, animal and plant secretions. In addition, synthetic fluids or polymer-based solutions that are analogs of biological fluids can be testing using this protocol to understand material properties under varying stresses, oscillation frequencies, and temperature. The protocol is well-suited for rheological characterization of biological fluids when very small sample volumes are made available.
The extended outcome of the protocol is that the apparent viscosity and apparent yield stress of GR mucus will facilitate the creation of analytical models to interpret results from fundamental hydrodynamic investigation of filter feeding and advance technologies requiring and involving crossflow and membrane filtration.
The macro-rheological study posits that the mucus in contact with food particulates is initially, in a gel-like state that serves as an adhesive. Upon initiation of flow and shear forces the mucus attains an apparent yield stress and undergoes plastic deformation. The protocol execution using a rotational rheometer helped in characterization of the transition of mucus from gel-like to fluid-like behavior. This transition was experimentally observed, and the apparent yield stress was recorded at 0.2736 Pa in rotational rheometer experiments. When the external stresses on the mucus are less than the apparent yield stress, the mucus will demonstrate gel-like behavior to facilitate adhesion of food particulates. When the external stresses exceed the apparent yield stress, mucus will demonstrate shear-thinning behavior that will facilitate transport of agglomerated food particulates to the digestive organs in the silver carp.

The silver carp, Hypophthalmichthys molitrix, is a planktivorous filter feeder and an invasive species that has infiltrated several natural waterways in the United States. This species was initially introduced in the upper Mississippi River basin to control algal blooms1,2,3. The silver carp is an extremely efficient feeder. Typically, its consumable food particle sizes range from 4 to 20 &#956;m to larger zooplankton that are around 80 &#956;m3,4,5. This species has outcompeted other native fish and can potentially cause enormous damage to native waterways by limiting available resources1,2,6. Thus, filter feeding fish such as the silver carp and the bighead carp pose a major threat to the Great Lakes1,2,6,7,8.
Filter feeding fish possess special organs called the gill rakers (GRs) with a thin layer of mucus residing on their surface. These organs improve the efficiency of filtration and aggregation of small particles from the incoming fluid. The goal of the protocol presented herein is to characterize the non-Newtonian, shear thinning material property and yield stress of the GR mucus acquired from the inner surface of the gill rakers in the silver carp. The value of yield stress of the GR-mucus, ascertained using a rotational rheometer, is of interest in this study. The measured yield stress also called the &#8220;apparent yield stress&#8221; depends on the testing methods such as steady shear rate- or dynamic oscillatory strain-type9,10. The shear-thinning, &#8216;yield-stress fluid,&#8217; undergoes a transition from solid-like to liquid-like behavior at a critical applied stress9,11. The apparent yield stress is the minimum shear stress required to initiate flow or that at which irreversible plastic deformation is first observed when the mucus transitions from a gel-like material to a fluid-like material. This behavior can be observed in structured viscoelastic materials. The transition from gel-like to fluid-like behavior of the GR mucus entails two functions i.e., an adhesive role to gather food particulates and a transport vehicle role to assist in the particulate delivery and filtration process. The extended function of the mucus includes creating diffusion barriers in disease resistance and respiration, providing controlled release of nutritional factors, toxic components and excretion, creating metabolic pathways for feeding and nesting, helping in predator protection, and producing boundary layer modifications that improve the locomotion and propulsive efficiency12,13,14.
Unlike simple fluids, complex fluids like the mucus possess properties that vary with flow conditions and require additional measurement parameters to define their bulk scale physical behavior. To monitor the viscosity and yield stress of GR mucus, rheological measurements are performed using a rotational rheometer. The rotational rheometer applies a steady or oscillatory shear stress or strain by means of a rotating disk in contact with the fluid sample and measures its response. The rationale behind using this instrument and technique is that the rheometer can provide a set of measurements to describe the material properties of the GR mucus of the silver carp, which cannot be defined by viscosity alone.
The mucus is a viscoelastic material and its mechanical response to an imposed deformation is between that of a pure solid (governed by Hooke&#8217;s law of elasticity) and that of a pure liquid (governed by Newton&#8217;s law of viscosity)15,16. The complex macromolecular network contained within the mucus can stretch and reorient in response to external forces or deformation. A rotational rheometer is comprised of a cone geometry and a Peltier plate as shown in Figure 1 and Figure 2 (see Table 1&#160; for instrumentation specifications). The objective of this study was to&#160;develop a protocol&#160;to determine the rheological properties of the GR mucus. An advantage of the rotational rheometer over a viscometer is its ability to make dynamic measurements using small sample volumes. The GR mucus sample volume in this study was approximately 1.4 mL. The viscometer, on the other hand, is limited to constant shear rates and requires large sample volumes.
The rheological properties of the mucus are expected to vary greatly within the silver carp anatomy. For example, the properties of the mucus residing on the GR surfaces may be different from the epibranchial organ. To account for the potential variability of mucus properties in different regions of the fish, the acquired GR mucus sample was diluted, and solutions of three concentrations were created and tested using the rotational rheometer.. The data and results regarding mucus rheology reported after executing the protocol demonstrated the efficacy of the measurement technique. The illustrative data presented in this paper are not meant to be generalized across the entire silver carp population. The protocol presented herein can be extended to investigate mucus rheology across larger sample sets to test other hypotheses.
The purpose of this study is to demonstrate the variation of rheological properties of GR mucus rheology with three different mucus concentrations (400 mg/mL, 200 mg/mL and 100 mg/mL). The 400 mg/mL concentration represents the raw mucus sample harvested from the fish GRs. Deionized water (DI) was used to dilute the raw mucus sample into 200 mg/mL and 100 mg/mL concentrations. Diluting the mucus samples allowed for the evaluation of the degree of shear thinning and apparent yield stress as a function of concentration and the determination of the &#160;concentration at which the GR mucus transitions to non-Newtonian behavior. A shaker was used to break down any large clumps of mucus in the samples to mitigate errors in the rheological data due to inhomogeneity.
In most vertebrates, including fish, the predominant mucus-forming macromolecules are glycoproteins (mucins) that tend to swell in water by entanglements or chemical cross-linking and create a gel-like material12,13,17,18,19,20. The high-molecular-weight, gel-forming macromolecules and high-water content reflects the slipperiness in the mucus13. A high degree of inter-macromolecular interactions leads to gel-formation whereas lower levels of inter-macromolecular interactions or broken bonds result in high-viscosity fluids21.
The processes of food particulate filtration in filter feeding fish are aided by GR mucus-related properties such as cohesion and viscosity that determine its potential for adhesion and tack22. The strength of mucus-based adhesion depends on specific intermolecular, electrostatic or hydrophobic interactions23. Sanderson et al.24 conducted a suspension-feeding study in blackfish wherein they found the evidence for mucus-based adhesion. They stated that the adhesion of suspended food particulates with a mucosal surface is followed by the transport of aggregated clumps of particles bound together with mucus by directed water-flow acting on it24. The mucus exposed to shear strain rates generated from water-flow facilitates the delivery of food particulates to digestive organs. Endoscopic techniques were used to observe filtered particles24.
Literature on the range of shear rates and practical limits in the rheological testing of GR mucus is scarce. Therefore, guidance was sought from rheological studies on gastric, nasal, cervical and lung mucus, salmon skin mucus, hagfish slime, and bone-joint surface lubricant wherein the rheological characterization and non-Newtonian attributes were studied11,12,25,26,27,28,29,30,31. More recently, the effect of fish skin mucus on locomotion and propulsive efficiency has been studied using constant shear rate viscometry. Skin mucus rheology studies (without any dilution or homogenization) pertaining to seabream, sea bass and meagre demonstrated non-Newtonian behavior at typically low shear rates14.&#160; In another related study, the raw skin mucus samples from dorsal and ventral sides of the Senegalese sole were found to exhibit non-Newtonian behavior, indicating a higher viscosity of the ventral mucus at all shear rates considered32. Other rheological protocols pertaining to the hydrogel scaffold development and for highly concentrated suspensions using a constant shear rate viscometer have also been reported in the literature33,34.
In this study, the GR mucus properties were investigated using a strain rate controlled, rotational rheometer that has been widely used in rheology experiments on complex biological fluids25. For Newtonian fluids, the apparent viscosity remains constant, is shear-rate-independent and the shear stresses vary linearly with shear strain rates (Figure 3A, B). For non-Newtonian fluids (such as shear-thinning fluids) viscosity is shear-rate-dependent or deformation-history-dependent (Figure 3A, B). The loss modulus (G&#8221;) represents the extent to which the material resists the tendency to flow and is representative of fluid viscosity (Figure 4). The storage modulus (G&#8217;) represents the tendency of the material to recover its original shape following stress-induced deformation and is equivalent to elasticity (Figure 4). The phase angle (&#948;) or loss tangent value, is calculated from the inverse tangent of G&#8221;/G&#8217;. It represents the balance between energy loss and storage and is also a common parameter for characterizing viscoelastic materials (&#948; = 0&#176; for a Hookean solid; &#948; = 90&#176; for a viscous liquid; &#948; &#60; 45&#176; for a viscoelastic solid and &#948; &#62; 45&#176; for a viscoelastic liquid) (Figure 4)25. The apparent yield stress (&#963;y) in structured fluids represents a change of state that can be observed in rheological data from steady state sweep and dynamic stress-strain sweeps10. If the external applied stress is less than the apparent yield stress, the material will deform elastically. When the stress exceeds the apparent yield stress (marked as &#8220;average stress&#8221; in Figure 3B), the material will transition from elastic to plastic deformation and begin to flow in its liquid state35. Measuring the storage modulus (G&#8217;) and loss modulus (G&#8221;) in the mucus-sample under oscillatory stress (or strain) conditions quantifies the change in the material state from gel-like to viscoelastic liquid-like behavior.
The types of rheometer tests performed to monitor data pertaining to&#160; storage modulus (G&#8217;), loss modulus (G&#8221;) and apparent viscosity (&#951;) are described here. The dynamic oscillation tests (strain sweeps and frequency sweeps) monitored G&#8217; and G&#8221; under controlled oscillation of cone geometry. The dynamic strain sweep tests determined the linear viscoelastic region (LVR) of the mucus by monitoring the intrinsic material response (Figure 4). Strain sweeps were used to determine the yielding behavior at constant oscillation frequency and temperature. The dynamic frequency sweep tests monitored the material response to increasing frequency (rate of deformation) at a constant amplitude (strain or stress) and temperature. Strain was maintained in the linear viscoelastic region (LVR) for the dynamic frequency sweep tests. The steady-state shear rate tests monitored the apparent viscosity (&#951;) under steady rotation of the cone geometry. The GR mucus was subjected to incremental stress steps and apparent viscosity (&#951;, Pa.s) was monitored for varying shear rate (&#253;, 1/s).
The protocol presented in this paper treats the GR mucus as a complex structured material of unknown viscoelasticity with a certain linear viscoelastic response range. The fish mucus was extracted from the GRs of the silver carp during a fishing expedition at the Hart creek location in the Missouri River&#160;by Professor L. Patricia Hernandez (Department of Biological Sciences, The George Washington University)&#160;1,2,36.&#160; An array of GRs inside the mouth of a Silver carp is shown in Figure 5A and a schematic drawing is presented in Figure 5B. An excised GR is shown in Figure 5C.&#160; The extraction of mucus from GRs of the silver carp is presented as an example in the schematic drawings, Figure 5D, E. All the rheometer tests were performed under a constant, controlled temperature of 22 &#177; 0.002 &#176;C, the temperature recorded at the fishing site1,2,36. &#160;Each mucus sample was tested three times with the rheometer, and the averaged results are presented along with the statistical error bars.

<table><tbody><tr><td>&lt;strong&gt;Materials&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Kim Wipes</td><td>VWR</td><td>470224-038</td><td>To clean Sample from plate</td></tr><tr><td>Gloves</td><td>VWR</td><td>89428-750</td><td>To prevent contamination of sample</td></tr><tr><td>Pipette</td><td>VWR</td><td>89079-974</td><td>To transport sample from vial to rheometer</td></tr><tr><td>Pipette Tips</td><td>Thermo Scientific</td><td>72830-042</td><td>To transport sample from vial to rheometer</td></tr><tr><td>Shaker</td><td>VWR</td><td>89032-094</td><td>To homogenously mix sample of mucus</td></tr><tr><td>Vials</td><td>VWR</td><td>66008-710</td><td>Contains measured sample volumes</td></tr><tr><td>Weigh Scale</td><td>Ohaus</td><td>Scout &amp;ndash;SPX Balances</td><td>To weigh mass of mucus samples</td></tr><tr><td>&lt;strong&gt;Chemical Reagents&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>De-Ionized Water (H20)</td><td>-</td><td>-</td><td>Liquid</td></tr><tr><td>Sterile 70% Isopropanol (C3H8O)</td><td>VWR</td><td>89108-162</td><td>Liquid</td></tr><tr><td>&lt;strong&gt;GR Mucus&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>100 mg/mL concentration, 2mL</td><td>-</td><td>-</td><td>Viscoelastic Material</td></tr><tr><td>400 mg/mL concentration, 1mL</td><td>-</td><td>-</td><td>Viscoelastic Material</td></tr><tr><td>200 mg/mL concentration, 1mL</td><td>-</td><td>-</td><td>Viscoelastic Material</td></tr><tr><td>&lt;strong&gt;Software&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>MATLAB</td><td>Mathworks</td><td>R2017a</td><td>Data analysis, post-processing and graphical representation</td></tr><tr><td>Trios</td><td>TA Instruments</td><td>v4.5.042498</td><td>Rheometer instrument control and analysis software</td></tr></tbody></table>

macro-rheology characterization of gill raker mucus in the silver carp, hypophthalmichthys molitrix

The silver carp, Hypophthalmichthys molitrix, is an invasive planktivorous filter feeder fish that infested the natural waterways of the upper Mississippi River basin due to its highly efficient filter feeding mechanism. The characteristic organs called gill rakers (GRs), found in many such filter feeders, facilitate the efficient filtration of food particles such as phytoplankton that are of a few microns in size.
The motivation to investigate the rheology of the GR mucus stems from our desire to understand its role in aiding the filter feeding process in the silver carp. The mucus-rich fluid, in a &#8216;thick and sticky&#8217; state may facilitate the adhesion of food particulates. The permeation and transport through the GR membrane are&#160;facilitated by the action of external shear forces that induce varying shear strain rates. Therefore, mucus rheology can provide a vital clue to the tremendous outcompeting nature of the silver carp within the pool of filter feeding fish. Based on this it was posited that GR mucus may provide an adhesive function to food particles and act as a transport vehicle to assist in the filter feeding process.
The main objective of the protocol is to determine the yield stress of the mucus, attributed to the minimum shear stress required to initiate flow at which irreversible plastic deformation is first observed across a structured viscoelastic material. Accordingly, rheological properties of the GR mucus, i.e., viscosity, storage, and loss moduli, were investigated for its non-Newtonian, shear-thinning nature using a rotational rheometer. &#160;
A protocol presented here is employed to analyze the rheological properties of mucus extracted from the gill rakers of a silver carp, fished at Hart Creek location of the Missouri River. The protocol aims to develop an effective strategy for rheological testing and material characterization of mucus assumed to be a structured viscoelastic material.

Watch this Scientific Journal Video about Macro-Rheology Characterization of Gill Raker Mucus in the Silver Carp, Hypophthalmichthys molitrix at JoVE.com

Macro-Rheology Characterization of Gill Raker Mucus in the Silver Carp, Hypophthalmichthys molitrix

This protocol presents a method to perform rheology characterization of mucus that resides on gill rakers (GRs) of the silver carp. Viscoelastic characteristics of GR-mucus, obtained by measuring viscosity, storage and loss moduli, are evaluated for the apparent yield stress to understand the filter feeding mechanism in GRs.

Macro-Rheology Characterization of Gill Raker Mucus in the Silver Carp, Hypophthalmichthys molitrix

The silver carp, Hypophthalmichthys molitrix, is an invasive planktivorous filter feeder fish that infested the natural waterways of the upper Mississippi ...

macro-rheology-characterization-gill-raker-mucus-silver-carp

Nanyang Technological University

Research

JoVE Journal

Engineering

3.0K Views.  The George Washington University. Dit protocol presenteert een methode om reologiekarakterisering uit te voeren van slijm dat zich op kieuwharken (GR's) van de zilverkarper bevindt. Visco-elastische kenmerken van GR-slijm, verkregen door het meten van viscositeit, opslag en verliesmodulus, worden geëvalueerd voor de schijnbare opbrengstspanning om het filtertoevoermechanisme in GR's te begrijpen.

Video: Macro-Rheology Characterization of Gill Raker Mucus in the Silver Carp, Hypophthalmichthys molitrix

A Time Differential Staining Technique Coupled with Full Bilateral Gill Denervation to Study Ionocytes in Fish

Branchial ionocytes (ICs) are the functional units for ionic regulation in fish. In adults, they are found on the filamental and lamellar epithelia of the gill where they transport ions such as Na+, Cl- and Ca2+ via a variety of ion channels, pumps and exchangers. The teleost gill is extrinsically innervated by the facial (VI), glossopharyngeal (IX) and vagus (X) nerves. The IX and X nerves are also the extrinsic source of branchial IC innervation. Here, two techniques used to study the innervation, proliferation and distribution of ICs are described: a time differential staining technique and a full bilateral gill denervation technique. Briefly, goldfish are exposed to a vital mitochondrion-specific dye (e.g., MitoTracker Red) which labels (red fluorescence) pre-existing ICs. Fish were either allowed to recover for 3 - 5 days or immediately underwent a full bilateral gill denervation. After 3 - 5 days of recovery, the gills are harvested and fixed for immunohistochemistry. The tissue is then stained with an &#945;-5 primary antibody (targets Na+/K+ ATPase containing cells) in conjunction with a secondary antibody that labels all (both new and pre-existing) ICs green. Using confocal imaging, it was demonstrated that pre-existing ICs appear yellow (labelled with both a viable mitochondrion-specific dye and &#945;-5) and new ICs appear green (labelled with &#945;-5 only). Both techniques used in tandem can be applied to study the innervation, proliferation and distribution of ICs on the gill filament when fish are exposed to environmental challenges.

This manuscript describes a protocol to track the re-distribution of branchial ionocytes and their innervation using a time differential staining technique coupled with full bilateral gill denervation.

Branchial ionocytes (ICs) are the functional units for ionic regulation in fish. In adults, they are found on the filamental and lamellar epithelia of the ...

Developmental Biology

VacuSIP, an Improved InEx Method for In Situ Measurement of Particulate and Dissolved Compounds Processed by Active Suspension Feeders

Benthic suspension feeders play essential roles in the functioning of marine ecosystems. By filtering large volumes of water, removing plankton and detritus, and excreting particulate and dissolved compounds, they serve as important agents for benthic-pelagic coupling. Accurately measuring the compounds removed and excreted by suspension feeders (such as sponges, ascidians, polychaetes, bivalves) is crucial for the study of their physiology, metabolism, and feeding ecology, and is fundamental to determine the ecological relevance of the nutrient fluxes mediated by these organisms. However, the assessment of the rate by which suspension feeders process particulate and dissolved compounds in nature is restricted by the limitations of the currently available methodologies. Our goal was to develop a simple, reliable, and non-intrusive method that would allow clean and controlled water sampling from a specific point, such as the excurrent aperture of benthic suspension feeders, in situ. Our method allows simultaneous sampling of inhaled and exhaled water of the studied organism by using minute tubes installed on a custom-built manipulator device and carefully positioned inside the exhalant orifice of the sampled organism. Piercing a septum on the collecting vessel with a syringe needle attached to the distal end of each tube allows the external pressure to slowly force the sampled water into the vessel through the sampling tube. The slow and controlled sampling rate allows integrating the inherent patchiness in the water while ensuring contamination free sampling. We provide recommendations for the most suitable filtering devices, collection vessel, and storing procedures for the analyses of different particulate and dissolved compounds. The VacuSIP system offers a reliable method for the quantification of undisturbed suspension feeder metabolism in natural conditions that is cheap and easy to learn and apply to assess the physiology and functional role of filter feeders in different ecosystems.

VacuSIP, an Improved InEx Method for In Situ Measurement of Particulate and Dissolved Compounds Processed by Active Suspension Feeders

We introduce the VacuSIP, a simple, non-intrusive, and reliable method for clean and accurate point sampling of water. The system was developed and evaluated for the simultaneous collection of the water inhaled and exhaled by benthic suspension feeders in situ, to cleanly measure removal and excretion of particulate and dissolved compounds.

Benthic suspension feeders play essential roles in the functioning of marine ecosystems. By filtering large volumes of water, removing plankton and ...

Environment

Salinity-dependent Toxicity Assay of Silver Nanocolloids Using Medaka Eggs

Salinity is an important characteristic of the aquatic environment. For aquatic organisms it defines the habitats of freshwater, brackish water, and seawater. Tests of the toxicity of chemicals and assessments of their ecological risks to aquatic organisms are frequently performed in freshwater, but the toxicity of chemicals to aquatic organisms depends on pH, temperature, and salinity. There is no method, however, for testing the salinity dependence of toxicity to aquatic organisms. Here, we used medaka (Oryzias latipes) because they can adapt to freshwater, brackish water, and seawater. Different concentrations of embryo-rearing medium (ERM) (1x, 5x, 10x, 15x, 20x, and 30x) were employed to test the toxicity of silver nanocolloidal particles (SNCs) to medaka eggs (1x ERM and 30x ERM have osmotic pressures equivalent to freshwater and seawater, respectively). In six-well plastic plates, 15 medaka eggs in triplicate were exposed to SNCs at 10 mg/L&#8722;1 in different concentrations of ERM at pH 7 and 25 &#176;C in the dark.
We used a dissecting microscope and a micrometer to measure heart rate per 15 sec and eye diameter on day 6 and full body length of the larvae on hatching day (section 4). The embryos were observed until hatching or day 14; we then counted the hatching rate every day for 14 days (section 4). To see silver accumulation in embryos, we used inductively coupled plasma mass spectrometry to measure the silver concentration of test solutions (section 5) and dechorionated embryos (section 6).The toxicity of the SNCs to medaka embryos obviously increased with increasing salinity. This new method allows us to test the toxicity of chemicals in different salinities.

Embryonic stages are the most susceptible to xenobiotics. Although chemical toxicity depends on salinity, no method exists to test the salinity dependence of toxicity to aquatic organisms. Here, we describe a new and high-throughput method for determining the salinity dependence of toxicity to aquatic embryos.

Salinity is an important characteristic of the aquatic environment. For aquatic organisms it defines the habitats of freshwater, brackish water, and ...

Challenges in Rheological Characterization of Highly Concentrated Suspensions &#8212; A Case Study for Screen-printing Silver Pastes

A comprehensive rheological characterization of highly concentrated suspensions or pastes is mandatory for a targeted product development meeting the manifold requirements during processing and application of such complex fluids. In this investigation, measuring protocols for a conclusive assessment of different process relevant rheological parameters have been evaluated. This includes the determination of yield stress, viscosity, wall slip velocity, structural recovery after large deformation and elongation at break as well as tensile force during filament stretching.
The importance of concomitant video recordings during parallel-plate rotational rheometry for a significant determination of rheological quantities is demonstrated. The deformation profile and flow field at the sample edge can be determined using appropriate markers. Thus, measurement parameter settings and plate roughness values can be identified for which yield stress and viscosity measurements are possible. Slip velocity can be measured directly and measuring conditions at which plug flow, shear banding or sample spillover occur can be identified clearly. 
Video recordings further confirm that the change in shear moduli observed during three stage oscillatory shear tests with small deformation amplitude in stage I and III but large oscillation amplitude in stage II can be directly attributed to structural break down and recovery. For the pastes investigated here, the degree of irreversible, shear-induced structural change increases with increasing deformation amplitude in stage II until a saturation is reached at deformations corresponding to the crossover of G' and G'', but the irreversible damage is independent of the duration of large amplitude shear. 
A capillary breakup elongational rheometer and a tensile tester have been used to characterize deformation and breakup behavior of highly filled pastes in uniaxial elongation. Significant differences were observed in all experiments described above for two commercial screen-printing silver pastes used for front side metallization of Si-solar cells.

A protocol for a robust and application relevant rheological characterization of highly concentrated suspensions is presented. Silver pastes used for screen-printing application in solar cell production are employed as model systems.

A comprehensive rheological characterization of highly concentrated suspensions or pastes is mandatory for a targeted product development meeting the ...

Characterization Of Multi-layered Fish Scales (Atractosteus spatula) Using Nanoindentation, X-ray CT, FTIR, and SEM

The hierarchical architecture of protective biological materials such as mineralized fish scales, gastropod shells, ram&#8217;s horn, antlers, and turtle shells provides unique design principles with potentials for guiding the design of protective materials and systems in the future. Understanding the structure-property relationships for these material systems at the microscale and nanoscale where failure initiates is essential. Currently, experimental techniques such as nanoindentation, X-ray CT, and SEM provide researchers with a way to correlate the mechanical behavior with hierarchical microstructures of these material systems1-6. However, a well-defined standard procedure for specimen preparation of mineralized biomaterials is not currently available. In this study, the methods for probing spatially correlated chemical, structural, and mechanical properties of the multilayered scale of A. spatula using nanoindentation, FTIR, SEM, with energy-dispersive X-ray (EDX) microanalysis, and X-ray CT are presented.

Characterization Of Multi-layered Fish Scales Atractosteus spatula Using Nanoindentation, X-ray CT, FTIR, and SEM

This paper presents the methods used for probing spatially correlated chemical, structural, and mechanical properties of the multilayered scale of Atractosteus spatula (A. spatula) using nanoindentation, Fourier transform infrared (FTIR) spectroscopy, scanning electron microscopy (SEM), and X-ray computed tomography (X-ray CT). The experimental results have been used to investigate the design principles of protective biological materials.

The hierarchical architecture of protective biological materials such as mineralized fish scales, gastropod shells, ram&#8217;s horn, antlers, and turtle ...

Bioengineering

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 ...

Combining Microfluidics and Microrheology to Determine Rheological Properties of Soft Matter during Repeated Phase Transitions

The microstructure of soft matter directly impacts macroscopic rheological properties and can be changed by factors including colloidal rearrangement during previous phase changes and applied shear. To determine the extent of these changes, we have developed a microfluidic device that enables repeated phase transitions induced by exchange of the surrounding fluid and microrheological characterization while limiting shear on the sample. This technique is &#181;2rheology, the combination of microfluidics and microrheology. The microfluidic device is a two-layer design with symmetric inlet streams entering a sample chamber that traps the gel sample in place during fluid exchange. Suction can be applied far away from the sample chamber to pull fluids into the sample chamber. Material rheological properties are characterized using multiple particle tracking microrheology (MPT). In MPT, fluorescent probe particles are embedded into the material and the Brownian motion of the probes is recorded using video microscopy. The movement of the particles is tracked and the mean-squared displacement (MSD) is calculated. The MSD is related to macroscopic rheological properties, using the Generalized Stokes-Einstein Relation. The phase of the material is identified by comparison to the critical relaxation exponent, determined using time-cure superposition. Measurements of a fibrous colloidal gel illustrate the utility of the technique. This gel has a delicate structure that can be irreversibly changed when shear is applied. &#181;2rheology data shows that the material repeatedly equilibrates to the same rheological properties after each phase transition, indicating that phase transitions do not play a role in microstructural changes. To determine the role of shear, samples can be sheared prior to injection into our microfluidic device. &#181;2rheology is a widely applicable technique for the characterization of soft matter enabling the determination of rheological properties of delicate microstructures in a single sample during phase transitions in response to repeated changes in the surrounding environmental conditions.

We demonstrate the fabrication and use of a microfluidic device that enables multiple particle tracking microrheology measurements to study the rheological effects of repeated phase transitions on soft matter.

The microstructure of soft matter directly impacts macroscopic rheological properties and can be changed by factors including colloidal rearrangement ...

Automatic Image Processing to Determine the Community Size Structure of Riverine Macroinvertebrates

Body size is an important functional trait that can be used as a bioindicator to assess the impacts of perturbations in natural communities. Community size structure responds to biotic and abiotic gradients, including anthropogenic perturbations across taxa and ecosystems. However, the manual measurement of small-bodied organisms such as benthic macroinvertebrates (e.g., &gt;500 &#181;m to a few centimeters long) is time-consuming. To expedite the estimation of community size structure, here, we developed a protocol to semi-automatically measure the individual body size of preserved river macroinvertebrates, which are one of the most commonly used bioindicators for assessing the ecological status of freshwater ecosystems. This protocol is adapted from an existing methodology developed to scan marine mesozooplankton with a scanning system designed for water samples. The protocol consists of three main steps: (1) scanning subsamples (fine and coarse sample size fractions) of river macroinvertebrates and processing the digitized images to individualize each detected object in each image; (2) creating, evaluating, and validating a learning set through artificial intelligence to semi-automatically separate the individual images of macroinvertebrates from detritus and artifacts in the scanned samples; and (3) depicting the size structure of the macroinvertebrate communities. In addition to the protocol, this work includes the calibration results and enumerates several challenges and recommendations to adapt the procedure to macroinvertebrate samples and to consider for further improvements. Overall, the results support the use of the presented scanning system for the automatic body size measurement of river macroinvertebrates and suggest that the depiction of their size spectrum is a valuable tool for the rapid bioassessment of freshwater ecosystems.

The article is based on the creation of an adapted protocol to scan, detect, sort, and identify digitized objects corresponding to benthic river macroinvertebrates using a semi-automatic imaging procedure. This procedure allows the acquisition of the individual size distributions and size metrics of a macroinvertebrate community in about 1 h.

Body size is an important functional trait that can be used as a bioindicator to assess the impacts of perturbations in natural communities. Community ...

Removing Zebrafish Scales for Culturing and Bone Biology Studies

In Vitro Culturing Technique for Studying Cellular Dynamics in Zebrafish Scales

Zebrafish scales offer a variety of advantages for use in standard laboratories for teaching and research purposes. Scales are easily collected without the need for euthanasia, regenerate within a couple of weeks, and are translucent and small, allowing them to be viewed using a standard microscope. Zebrafish scales are especially useful in educational environments, as they provide a unique opportunity for students to engage in hands-on learning experiences, particularly in understanding cellular dynamics and in vitro culturing methods. The main objective of this protocol is to describe a method for collecting and maintaining zebrafish scales in culture for use in a variety of biological studies using basic laboratory equipment. Additionally, the protocol details their use in understanding bone homeostasis by examining the activity of bone cells involved in bone resorption and deposition. It also includes additional protocols for general techniques, such as the visualization of nuclei and apoptotic cells. The in vitro culturing protocol produces reliable results with minimal reagents and equipment. This article discusses the benefits of using in vitro cultures of zebrafish scales to foster scientific inquiry and outlines the resources needed to support their integration into educational settings.

Author Spotlight: Advancing Bone Cell Activity Research with Zebrafish Scales 

This protocol offers a method to study cellular dynamics using a simple in vitro culture technique. It provides an opportunity for zebrafish researchers and educators to study cellular processes, such as those related to bone homeostasis and basic cell biology, by visualizing fluorescent nuclei and apoptotic cells within the scales.

Zebrafish scales offer a variety of advantages for use in standard laboratories for teaching and research purposes. Scales are easily collected without ...

Rapid Viscoelastic Characterization of Airway Mucus Using a Benchtop Rheometer

In muco-obstructive lung diseases (e.g., asthma, chronic obstructive pulmonary disease, cystic fibrosis) and other respiratory conditions (e.g., viral/bacterial infections), mucus biophysical properties are altered by goblet cell hypersecretion, airway dehydration, oxidative stress, and the presence of extracellular DNA. Previous studies showed that sputum viscoelasticity correlated with pulmonary function and that treatments affecting sputum rheology (e.g., mucolytics) can result in remarkable clinical benefits. In general, rheological measurements of non-Newtonian fluids employ elaborate, time-consuming approaches (e.g., parallel/cone-plate rheometers and/or microbead particle tracking) that require extensive training to perform the assay and interpret the data. This study tested the reliability, reproducibility, and sensitivity of Rheomuco, a user-friendly benchtop device that is designed to perform rapid measurements using dynamic oscillation with a shear-strain sweep to provide linear viscoelastic moduli (G&#39;, G&#34;, G*, and tan &#948;) and gel point characteristics (&#947;c&#160;and &#963;c) for clinical samples within 5 min. Device performance was validated using different concentrations of a mucus simulant, 8 MDa polyethylene oxide (PEO), and against traditional bulk rheology measurements. A clinical isolate harvested from an intubated patient with status asthmaticus (SA) was then assessed in triplicate measurements and the coefficient of variation between measurements is &#60;10%. Ex vivo use of a potent mucus reducing agent, TCEP, on SA mucus resulted in a five-fold decrease in elastic modulus and a change toward a more &#34;liquid-like&#34; behavior overall (e.g., higher tan &#948;). Together, these results demonstrate that the tested benchtop rheometer can make reliable measures of mucus viscoelasticity in clinical and research settings. In summary, the described protocol could be used to explore the effects of mucoactive drugs (e.g., rhDNase, N-acetyl cysteine) onsite to adapt treatment on a case-by-case basis, or in preclinical studies of novel compounds.

The viscoelastic properties of mucus play a critical role in mucociliary clearance. However, traditional mucus rheological techniques require complex and time-consuming approaches. This study provides a detailed protocol for the use of a benchtop rheometer that can rapidly and reliably perform viscoelastic measurements.

Macro-Rheology Characterization of Gill Raker Mucus in the Silver Carp, Hypophthalmichthys molitrix

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