Name: macro-rheology characterization of gill raker mucus in the silver carp, hypophthalmichthys molitrix
Uploaded: 2020-07-10T14:00:00
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...

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	<li>Cohen, K. E., Hernandez, L. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+complex+trophic+anatomy+of+silver+carp,+Hypophthalmichthys+molitrix,+highlighting+a+novel+type+of+epibranchial+organ.">The complex trophic anatomy of silver carp, Hypophthalmichthys molitrix, highlighting a novel type of epibranchial organ.</a> Journal of Morphology. 279, 1615-1628 (2018).</li><li>Cohen, K. E., Hernandez, L. 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C., Jones, R., Reid, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Identification+of+glycoproteins+in+goblet+cells+of+epidermis+and+gill+of+plaice+(Pleuroneces+platessa+L.),+flounder+(Platichthys+flesus+(L.))+and+rainbow+trout+(Salmo+gairdneri+Richardson).">Identification of glycoproteins in goblet cells of epidermis and gill of plaice (Pleuroneces platessa L.), flounder (Platichthys flesus (L.)) and rainbow trout (Salmo gairdneri Richardson).</a> Histochemical Journal. 8, 597-608 (1976).</li><li>Silberberg, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mucus+glycoprotein,+its+biophysical+and+gel+forming+properties.">Mucus glycoprotein, its biophysical and gel forming properties.</a> Symposia of the Society for Experimental Biology. 43, 43-64 (1989).</li><li>Hills, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> The Biology of Surfactants. , 408 (1988).</li><li>Aubert, H., Brook, A. 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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. 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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. 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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 volum...

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.

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			<td height="21" style="height:21px;width:191px;">Materials</td>
			<td style="width:245px;"></td>
			<td style="width:191px;"></td>
			<td style="width:261px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:191px;">Kim Wipes</td>
			<td style="width:245px;">VWR</td>
			<td style="width:191px;">470224-038</td>
			<td style="width:261px;">To clean Sample from plate</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:191px;">Gloves</td>
			<td style="width:245px;">VWR</td>
			<td style="width:191px;">89428-750</td>
			<td style="width:261px;">To prevent contamination of sample</td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:191px;">Pipette</td>
			<td style="width:245px;">VWR</td>
			<td style="width:191px;">89079-974</td>
			<td style="width:261px;">To transport sample from vial to rheometer</td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:191px;">Pipette Tips</td>
			<td style="width:245px;">Thermo Scientific</td>
			<td style="width:191px;">72830-042</td>
			<td style="width:261px;">To transport sample from vial to rheometer</td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:191px;">Shaker</td>
			<td style="width:245px;">VWR</td>
			<td style="width:191px;">89032-094</td>
			<td style="width:261px;">To homogenously mix sample of mucus</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:191px;">Vials</td>
			<td style="width:245px;">VWR</td>
			<td style="width:191px;">66008-710</td>
			<td style="width:261px;">Contains measured sample volumes</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:191px;">Weigh Scale</td>
			<td style="width:245px;">Ohaus</td>
			<td style="width:191px;">Scout &#8211;SPX Balances</td>
			<td style="width:261px;">To weigh mass of mucus samples</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:191px;">Chemical Reagents</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:191px;">De-Ionized Water (H20)</td>
			<td style="width:245px;">-</td>
			<td style="width:191px;">-</td>
			<td style="width:261px;">Liquid</td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:191px;">Sterile 70% Isopropanol (C3H8O)</td>
			<td style="width:245px;">VWR</td>
			<td style="width:191px;">89108-162</td>
			<td style="width:261px;">Liquid</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">GR Mucus</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:191px;">100 mg/mL concentration, 2mL</td>
			<td style="width:245px;">-</td>
			<td style="width:191px;">-</td>
			<td style="width:261px;">Viscoelastic Material</td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:191px;">400 mg/mL concentration, 1mL</td>
			<td style="width:245px;">-</td>
			<td style="width:191px;">-</td>
			<td style="width:261px;">Viscoelastic Material</td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:191px;">200 mg/mL concentration, 1mL</td>
			<td style="width:245px;">-</td>
			<td style="width:191px;">-</td>
			<td style="width:261px;">Viscoelastic Material</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:191px;">Software</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:191px;">MATLAB</td>
			<td style="width:245px;">Mathworks</td>
			<td style="width:191px;">R2017a</td>
			<td style="width:261px;">Data analysis, post-processing and graphical representation</td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:191px;">Trios</td>
			<td>TA Instruments</td>
			<td style="width:191px;">v4.5.042498</td>
			<td style="width:261px;">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.

In this section, we present the results of the experiments on GR mucus using a rotational rheometer with a cone geometry (40 mm diameter, 1&#176; 0&#8217; 11&#8217;&#8217;) and a Peltier plate. The experiments helped in characterizing the non-Newtonian, shear-thinning behavior of the GR mucus and the apparent yield stress depicting the mucus transition from a gel-like material to a fluid-like material. The representative results entail quantitative descriptions of low-torque limits and secondary flow effects of the rotat...

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 ...

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