This work is supported by Multi-University Research Initiative through the Air Force Office of Scientific Research of the United States (AFOSR-FA9550-15-1-0009) (M. B. F., S. E. N., J.-Y. J., J. M). Collection of pink sea urchins was supported by the University of California Ship Funds and the US National Marine Fisheries Service (K.N.S., J.R.A.T). The authors acknowledge the following people: Prof. Jerry Tustaniwskyj for helpful suggestions during development of the bioinspired Aristotle's lantern sampler, Prof. Marc A. Meyers (UCSD, Dept. of Mechanical and Aerospace Engineering, Materials Science and Engineering Program), Prof. Robert L. Sah and Esther Cory (UCSD, Dept. of Bioengineering), and Dr. Maya deVries (Marine Biology Research Division, Scripps Institution of Oceanography). We also thank undergraduate students Sze Hei Siu, Jerry Ng and Ivan Torres for polishing urchin teeth cross-sections.

1. Biological Materials Science
<ol>
	<li>Wear personal protective equipment (i.e., gloves, safety glasses and lab coat) and follow all applicable safety procedures for using dissecting tools.</li>
	<li>Rinse off the forceps and scalpel with distilled water to use for dissection.</li>
	<li>Thaw a frozen pink sea urchin at RT for 1 hr. Place a thawed specimen in a glass dish with sufficient space to be able to maneuver the urchin and cutting tools. Turn the urchin upside down so that the teeth tips face up.
	<ol>
		<li>Cut away the connective tissue around the perimeter of the Aristotle&#39;s lantern with the scalpel and carefully lift out the lantern. Rinse the lantern off with running distilled water. Discard unused urchin parts in a proper waste disposal container.</li>
		<li>Turn the Aristotle&#39;s lantern over again so the tips of the teeth face down. Locate the plumula end of each tooth (opposite to the tip) facing up and use the forceps to carefully slide out individual teeth from the lantern.</li>
	</ol>
	</li>
	<li>Prepare epoxy to pot the teeth. Weigh 5 g of resin and add 1.15 g of hardener (e.g., 100 parts resin to 23 parts hardener by weight) in a shallow disposable plastic tray. Mix contents together slowly without forming bubbles. 
	Note: Do not leave leftover mixed epoxy in a container with insufficient exposure to the atmosphere. The curing process is exothermic and can ignite nearby flammables. Keep any leftover mixed epoxy in a well-ventilated fume hood away from flammable items.
	<ol>
		<li>Lubricate a 2.5 dram plastic tube (22 mm inner diameter, 39 mm length) using petroleum jelly applied with a finger and wipe off any excess with a tissue. Fill up the tube halfway with mixed epoxy.</li>
		<li>Use the forceps to pick up a tooth and carefully submerge it in the epoxy with the curved concave side facing up. Let the epoxy cure at RT for 24 hr. 
		Note: Prevent the tooth tip from drifting to touch the plastic tube wall as the epoxy cures since this will make polishing the tip more difficult.</li>
	</ol>
	</li>
	<li>Place the plastic tube with cured epoxy in a vise. Tighten the vise slowly until a fissure is made in the plastic tube. Peel away residual plastic from the epoxy surface.
	<ol>
		<li>Use a sectioning saw to cut the epoxy around the tooth down to a smaller block (1 cm3).</li>
	</ol>
	</li>
	<li>Prepare a clean area for polishing and set up a flat work station with a hard plastic board. Fill a squirt bottle with distilled water.
	<ol>
		<li>Start with the lowest grit sandpaper available (e.g., 120) and squeeze a small amount of water from the wash bottle onto the sandpaper. Using light pressure, rub the sample in one back and forth direction (e.g., left-right) for 5 min.</li>
		<li>Wash off the surface of the sample over a sink and wipe off with a particle-free tissue. Remove any leftover sandpaper grit with compressed air for 15 sec.</li>
		<li>Use progressively higher grit sandpaper (e.g., 600 and 2,400) to repeat protocol steps 1.6.1 and 1.6.2. Using light pressure, rub the sample in a back and forth direction perpendicular to the previous polish step (e.g., up-down, left-right). 
		Note: Use a light microscope at 20X magnification to see perpendicular scratch marks intersect with each grit level (e.g., 120, 600, 2,400). Move to the next higher grit sandpaper when scratch marks from the previous grit level disappear.</li>
		<li>Prepare a squirt bottle with 3-&#181;m diamond polishing suspension in a 1:1 distilled water solution. Use a polish cloth for diamond suspensions to repeat protocol steps 1.6.1 and 1.6.2.</li>
		<li>Prepare a squirt bottle with 0.5-&#181;m alumina polishing suspension in a 1:1 distilled water solution. Use a microcloth polishing surface to repeat protocol steps 1.6.1 and 1.6.2. 
		Note: Fine scratch marks from protocol steps 1.6.4 and 1.6.5 will not be visible at 20X magnification. For these protocol steps, polish for 5 min in a back and forth motion to remove all previous scratches.</li>
		<li>Clean the polished surface with distilled water and use particle-free tissue with compressed air to carefully dry. Wrap with particle-free tissue to maintain mirror polished finish. 
		Note: Dry all polishing surfaces face down on large particle-free tissues. Store in a plastic sleeve to avoid dust particles settling on the surface between polishing times.</li>
	</ol>
	</li>
	<li>Characterize the sea urchin tooth microstructure using scanning electron microscopy (SEM). Use a sputter coater to sputter iridium with a deposition current of 85 mA for 10 sec onto the polished tooth surface for a coating thickness of ~20 nm.
	<ol>
		<li>Obtain micrograph images at 250X - 4,000X magnification using a SEM. 
		Note: Use 5 kV in the scanning electron (SE) mode and 15 kV in the back-scattered electron (BSE) mode. Use BSE mode to identify calcite fibers interspersed with Mg-enriched polycrystalline matrix.</li>
	</ol>
	</li>
	<li>Perform micro-computed tomography (&#181;-CT) scans of a whole pink sea urchin and a freshly dissected Aristotle&#39;s lantern. Position each thawed sample inside the closed chamber container with a moistened tissue to provide a humidified environment while scanning.
	<ol>
		<li>Scan the whole urchin and the Aristotle&#39;s lantern by &#181;-CT with an isotropic voxel size of 36.00 &#181;m and 9.06 &#181;m, respectively. Apply an electrical potential of 100 kVp and 70 kVp with current of 100 mA and 141 mA, for the whole urchin and the Aristotle&#39;s lantern, respectively, using a 1.0 mm aluminum filter for both.</li>
		<li>Apply a beam hardening correction algorithm during image reconstruction to account for beam hardening artifacts that result from the &#181;-CT x-ray source emitting x-rays of multiple energies using manufacturer&#39;s protocol.</li>
	</ol>
	</li>
	<li>Use imaging software to refine image segmentation and acquire a triangle mesh model for the Aristotle&#39;s lantern structure.
	<ol>
		<li>Load and preview Aristotle&#39;s lantern image data from the &#181;-CT scan. Match the voxel size (9.06 &#181;m) to the values from the micro-CT scan.</li>
		<li>Use a volume rendering function to visualize the Aristotle&#39;s lantern in 3D space. Adjust the 2D orthogonal slice with the Bounding Box module and adjust the threshold value/color with the Volume Rendering module.</li>
		<li>Make mask segments for the region of interest (e.g., sea urchin tooth) using the segmentation editor. Select XY, YZ, and XZ planes and the 3D isometric view. Use the magic wand (black arrow) to distinguish between simple structures (tooth vs. pyramid) in the Aristotle&#39;s lantern.</li>
		<li>Reconstruct the model surface from the extracted mask segments. Select the Surface Generation module and apply. Deselect the Volume Rendering Settings to have the visible top surface disappear. Add the Surface View module to display the surface result.</li>
		<li>Simplify the model surface by reducing the number of faces to &#60; 18,000.</li>
		<li>Edit individual triangle mesh on the model surface as needed. Save the model as a stereolithography (STL) file for exporting to use with computer-assisted design (CAD) modeling software.</li>
	</ol>
	</li>
</ol>
2. Bioinspired Design
<ol>
	<li>Use the Aristotle&#39;s lantern from the micro-CT scan as a reference to make a bioinspired design with CAD modeling software. 
	Note: The bioinspired design has five curved teeth with height 6 cm and diameter 8 cm for the closed lantern. It is scaled up ~5x from the size of the natural Aristotle&#39;s lantern.</li>
	<li>Save the STL file parts to a flash drive and upload the files to a fused deposition modeling (FDM) 3D printer.
	<ol>
		<li>Load acrylonitrile butadiene styrene (ABS) plastic and support plastic material cartridges into the appropriate slots of the 3D printer.</li>
		<li>Insert the modeling base on the Z platform and align tabs with slots on the metal tray.</li>
		<li>Open each of the STL file parts and follow the display screen steps to print all the lantern parts at the same time. 
		Note: Lantern parts must fit within the building envelope (25 x 25 x 30 cm3) for the 3D printer. All five teeth are arranged on the modeling base and printed simultaneously with the tooth tip facing up. The build rate is 16 cm3 per hour and the total build time is approximately 8 hr.</li>
		<li>Release the modeling base from the tabs when all file parts are printed and slide the base out of the 3D printer along the tray guides.</li>
		<li>Use a metal spatula to pry all parts off the base and a metal file to wear down any extra plastic attached to the parts.</li>
		<li>Place the printed parts into a heated base bath until the support plastic material dissolves.</li>
	</ol>
	</li>
	<li>Fasten each tooth to a joint arm with a link rod and two E-retaining rings on either side. 
	Note: Refer to Figure 6 for assembly of the bioinspired Aristotle&#39;s lantern.</li>
</ol>
3. Bioexploration
<ol>
	<li>Use the CAD file for the bioinspired tooth to do a finite element modeling (FEM) stress analysis test.
	<ol>
		<li>Open the file (xx.sldprt) to do further engineering analysis. Above the &#34;Office Products&#34; tab, select the &#34;SolidWorks Simulation&#34; button.</li>
		<li>Above the &#34;Simulation&#34; tab, select the &#34;Study Advisor&#34; button and then the drop-down option &#34;New Study&#34;.</li>
		<li>Select the type of simulation test to be run by choosing &#34;Static&#34;.</li>
		<li>On the Static Test list, right-click on &#34;Fixtures&#34; and select &#34;Fixed Geometry&#34;.</li>
		<li>Click on the inner faces to add fixtures to the mounting holes where pins will go.</li>
		<li>On the Static Test list, right-click on &#34;External Loads&#34; and select &#34;Force&#34;.</li>
		<li>Click on the tooth grinding tip faces to apply 45 N force to the edges.</li>
		<li>On the Static Test list, right-click on &#34;External Loads&#34; and select &#34;Gravity&#34;.</li>
		<li>Indicate &#34;Top Plane&#34; for the gravity force applied normal to the plane.</li>
		<li>On the Static Test list, right-click on &#34;Mesh&#34; and select &#34;Create Mesh&#34;.</li>
		<li>Move the scale bar for &#34;Mesh Density&#34; all the way to the right for &#34;Fine&#34;.</li>
		<li>On the Static Test list, right-click on &#34;Static&#34; and select &#34;Run&#34; to run the test. 
		Note: The colored scale bar for areas of highest stress and the &#34;Yield Strength&#34;.</li>
	</ol>
	</li>
	<li>Compare stress analysis test results for the bioinspired tooth with and without the keel.</li>
</ol>

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</ol>

We have nothing to disclose.

Sea urchins use the Aristotle&#39;s lantern (Figure 1A) for a variety of functions (feeding, boring, pivoting, etc.). The fossil record indicates that the lantern has evolved in shape and function from the most primitive cidaroid type to the camarodont type of modern sea urchins14. Cidaroid lanterns have longitudinally grooved teeth (Figure 1B, top) and non-separated muscle attachment to its pyramid structure. This limits their up and down movement and robs them of th...

The fields of biology, biological materials science, biomaterials, bioengineering and biochemistry employ the premiere scientific techniques and minds in an attempt to provide a deeper understanding of the incredible natural world. This research has explained many of the most amazing biological structures and organisms; from the intrinsic toughness of human bone1,2 to the large beak of the toucan3. However, much of this knowledge is difficult to employ in a manner that can provide a benefit to society. As a result, the tangential field of bioinspiration employs the lessons learned from nature to modern materials in order to solve common problems. Examples include superhydrophobic surfaces inspired by lotus leaves4-6, adhesive surfaces inspired by the feet of geckos and insects7,8, tough ceramics inspired by the nacre of abalone9-11 and biopsy harvesters inspired by the mouthpiece of the sea urchin, also known as the Aristotle&#39;s lantern12,13.
Sea urchins are invertebrate animals covered with spines whose habitat most commonly consists of the rocky beds on the ocean floor. The body (called a test) in the largest urchin species can be more than 18 cm in diameter; test size in pink sea urchins (Strongylocentrotus fragilis) examined in this study can grow to 10 cm diameter. The Aristotle&#39;s lantern is composed of five predominately calcium carbonate teeth supported by pyramid structures composed of mineralized tissue and arranged into a dome-like formation that enclose all but the distal grinding tips of the teeth (Figure 1A).
The muscle structure of the jaws is capable of efficient chewing and scraping even against hard ocean rocks and corals. When the jaws open, the teeth protrude outwards and when the jaws close, the teeth retract inwards in a single smooth motion. Comparison between primitive (above) and modern (below) sea urchin tooth cross-sections (Figure 1B) indicates that a keeled tooth evolved to strengthen the tooth when grinding against hard substrates. Each individual tooth has a slightly convex curvature and a T-shaped morphology in the transverse plane (normal to the growth direction) due to the longitudinally attached keel (Figure 1C, D).
Bioinspiration begins with observation of interesting natural phenomena, such as the efficient chewing motion of the Aristotle&#39;s lantern in sea urchins. This natural structure initially captivated Aristotle because it reminded him of a horn lantern with the panes of horn left out. More than two millennia later, Scarpa was fascinated by the complexity of the Aristotle&#39;s lantern that he and later Trogu mimicked the natural chewing motion using only paper and rubber bands (Figure 2A)15,16. Similarly, Jelinek was bioinspired by the chewing motion of the Aristotle&#39;s lantern and developed a better biopsy harvester that could safely isolate tumorous tissue without spreading cancerous cells (Figure 2B, C)12,13. In this case, bioinspired design was utilized to make a biomedical device that fit a specific need for a desired application.
The design protocol described here applies to a sediment sampler bioinspired by sea urchins. Through biological materials science, the natural structure of the Aristotle&#39;s lantern is characterized. Bioinspired design identifies potential applications where the natural mechanisms can be enhanced through the use of modern materials and fabrication techniques. The final design is re-examined through the prism of bioexploration to understand how the natural tooth structure evolved (Figure 3). The last bioexploration step, proposed by Porter17,18, uses engineering analysis methods to explore and explain biological phenomena. All the important steps of the bioinspiration process are presented as an example for harnessing technology, pre-approved by nature, which can be used for solving modern problems. Our protocol, motivated by previous bioinspiration procedures presented for specific applications by Arzt7, is targeted for biologists, engineers and anyone else who is inspired by nature.

<table><tbody><tr><td>BUEHLERMET II 8 PLN 600/P1200</td><td>Buehler</td><td>305308600102</td><td>Abrasive paper for polishing</td></tr><tr><td>TRIDENT POLISH CLOTH 8&quot; PSA</td><td>Buehler</td><td>407518</td><td>Polish cloth for 3 &amp;#956;m suspension</td></tr><tr><td>METADI SUPREME POLY SUSP,3MIC</td><td>Buehler</td><td>406631</td><td>Polish suspension (3&amp;nbsp;&amp;#956;m)</td></tr><tr><td>MICROCLOTH FOR 8 IN WHEEL PSA</td><td>Buehler</td><td>407218</td><td>Polish cloth for 50 nm suspension</td></tr><tr><td>MASTERPREP SUSPENSION, 6 OZ</td><td>Buehler</td><td>636377006</td><td>Polish suspension (50&amp;nbsp;nm)</td></tr><tr><td>Skyscan 1076 micro-CT Scanner</td><td>Bruker</td><td></td><td>Micro-CT scanner equipment</td></tr><tr><td>Amira software</td><td>FEI Visualization Sciences Group</td><td></td><td>Software for 3D manipulation of Micro-CT scans</td></tr><tr><td>FEI Philips XL30</td><td>FEI Philips</td><td></td><td>ESEM equipment for characterization of polished tooth cross-sections</td></tr><tr><td>SolidWorks Design software</td><td>Dassault Systems</td><td></td><td>Design software for CAD drawing bioinspired device</td></tr><tr><td>SolidWorks Simulation software</td><td>Dassault Systems</td><td></td><td>Simulation software for stress test of CAD drawing bioinspired device</td></tr><tr><td>Dimension 1200es</td><td>Stratasys</td><td></td><td>3D printer for fabrication of bioinspired device from CAD drawing</td></tr><tr><td>ABSplus</td><td>Stratasys</td><td></td><td>3D printer plastic</td></tr></tbody></table>

a protocol for bioinspired design: a ground sampler based on sea urchin jaws

Bioinspired design is an emerging field that takes inspiration from nature to develop high-performance materials and devices. The sea urchin mouthpiece, known as the Aristotle's lantern, is a compelling source of bioinspiration with an intricate network of musculature and calcareous teeth that can scrape, cut, chew food and bore holes into rocky substrates. We describe the bioinspiration process as including animal observation, specimen characterization, device fabrication and mechanism bioexploration. The last step of bioexploration allows for a deeper understanding of the initial biology. The design architecture of the Aristotle's lantern is analyzed with micro-computed tomography and individual teeth are examined with scanning electron microscopy to identify the microstructure. Bioinspired designs are fabricated with a 3D printer, assembled and tested to determine the most efficient lantern opening and closing mechanism. Teeth from the bioinspired lantern design are bioexplored via finite element analysis to explain from a mechanical perspective why keeled tooth structures evolved in the modern sea urchins we observed. This circular approach allows for new conclusions to be drawn from biology and nature.

Bioinspired design of the Aristotle's lantern sampling device depends heavily upon the quality of the characterization methods used. Non-invasive techniques like µ-CT are helpful for analyzing the whole lantern and individual teeth to apply application specific enhancements for the bioinspired design (Figure 4). Meanwhile, the tooth microstructure can be explored via secondary electron and back-scattered electron micrographs of the polished cross-section of an individual...

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A Protocol for Bioinspired Design: A Ground Sampler Based on Sea Urchin Jaws

A protocol for bioinspired design is described for a sampling device based on the jaws of a sea urchin. The bioinspiration process includes observing the sea urchins, characterizing the mouthpiece, 3D printing of the teeth and their assembly, and bioexploring the tooth structure.

Bioinspired design is an emerging field that takes inspiration from nature to develop high-performance materials and devices. The sea urchin mouthpiece, ...

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Research

JoVE Journal

Bioengineering

11.0K Views.  University of California, San Diego. A protocol for bioinspired design is described for a sampling device based on the jaws of a sea urchin. The bioinspiration process includes observing the sea urchins, characterizing the mouthpiece, 3D printing of the teeth and their assembly, and bioexploring the tooth structure. 

Video: A Protocol for Bioinspired Design: A Ground Sampler Based on Sea Urchin Jaws

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

Protocol for Microplastics Sampling on the Sea Surface and Sample Analysis

Microplastic pollution in the marine environment is a scientific topic that has received increasing attention over the last decade. The majority of scientific publications address microplastic pollution of the sea surface. The protocol below describes the methodology for sampling, sample preparation, separation and chemical identification of microplastic particles. A manta net fixed on an &#187;A frame&#171; attached to the side of the vessel was used for sampling. Microplastic particles caught in the cod end of the net were separated from samples by visual identification and use of stereomicroscopes. Particles were analyzed for their size using an image analysis program and for their chemical structure using ATR-FTIR and micro FTIR spectroscopy. The described protocol is in line with recommendations for microplastics monitoring published by the Marine Strategy Framework Directive (MSFD) Technical Subgroup on Marine Litter. This written protocol with video guide will support the work of researchers that deal with microplastics monitoring all over the world.

The protocol below describes the methodology for: microplastics sampling on the sea surface, separation of microplastic and chemical identification of particles. This protocol is in line with the recommendations for microplastics monitoring published by the MSFD Technical Subgroup on Marine Litter.

Microplastic pollution in the marine environment is a scientific topic that has received increasing attention over the last decade. The majority of ...

Building Finite Element Models to Investigate Zebrafish Jaw Biomechanics

Skeletal morphogenesis occurs through tightly regulated cell behaviors during development; many cell types alter their behavior in response to mechanical strain. Skeletal joints are subjected to dynamic mechanical loading. Finite element analysis (FEA) is a computational method, frequently used in engineering that can predict how a material or structure will respond to mechanical input. By dividing a whole system (in this case the zebrafish jaw skeleton) into a mesh of smaller 'finite elements', FEA can be used to calculate the mechanical response of the structure to external loads. The results can be visualized in many ways including as a 'heat map' showing the position of maximum and minimum principal strains (a positive principal strain indicates tension while a negative indicates compression. The maximum and minimum refer the largest and smallest strain). These can be used to identify which regions of the jaw and therefore which cells are likely to be under particularly high tensional or compressional loads during jaw movement and can therefore be used to identify relationships between mechanical strain and cell behavior. This protocol describes the steps to generate Finite Element models from confocal image data on the musculoskeletal system, using the zebrafish lower jaw as a practical example. The protocol leads the reader through a series of steps: 1) staining of the musculoskeletal components, 2) imaging the musculoskeletal components, 3) building a 3 dimensional (3D) surface, 4) generating a mesh of Finite Elements, 5) solving the FEA and finally 6) validating the results by comparison to real displacements seen in movements of the fish jaw.

Finite Element Analysis is a frequently used tool to investigate the mechanical performance of structures under load. Here we apply its use to modeling the biomechanics of the zebrafish jaw.

Skeletal morphogenesis occurs through tightly regulated cell behaviors during development; many cell types alter their behavior in response to mechanical ...

Developmental Biology

Bioindication Testing of Stream Environment Suitability for Young Freshwater Pearl Mussels Using In Situ Exposure Methods

Knowledge of habitat suitability for freshwater mussels is an important step in the conservation of this endangered species group. We describe a protocol for performing in situ juvenile exposure tests within oligotrophic river catchments over one-month and three-month periods. Two methods (in both modifications) are presented to evaluate the juvenile growth and survival rate. The methods and modifications differ in value for the locality bioindication and each has its benefits as well as limitations. The sandy cage method works with a large set of individuals, but only some of the individuals are measured and the results are evaluated in bulk. In the mesh cage method, the individuals are kept and measured separately, but a low individual number is evaluated. The open water exposure modification is relatively easy to apply; it shows the juvenile growth potential of sites and can also be effective for water toxicity testing. The within-bed exposure modification needs a high workload but is closer to the conditions of a natural juvenile environment and it is better for reporting the real suitability of localities. On the other hand, more replications are needed in this modification due to its high-hyporheic environment variability.

Bioindication Testing of Stream Environment Suitability for Young Freshwater Pearl Mussels Using In Situ Exposure Methods

In situ bioindications enable determination of the suitability of an environment for endangered mussel species. We describe two methods based on the juvenile exposure of freshwater pearl mussels in cages to oligotrophic river habitats. Both methods are implemented in variants for open water and hyporheic water environments.

Knowledge of habitat suitability for freshwater mussels is an important step in the conservation of this endangered species group. We describe a protocol ...

Methods for Image-based Surveys of Benthic Macroinvertebrates and Their Habitat Exemplified by the Drop Camera Survey for the Atlantic Sea Scallop

Underwater imaging has long been used in the field of marine ecology but decreasing costs of high-resolution cameras and data storage have made the approach more practical than in the past. Image-based surveys allow for initial samples to be revisited and are non-invasive compared to traditional survey methods that typically involve nets or dredges. Protocols for image-based surveys can vary greatly but should be driven by target species behavior and survey objectives. To demonstrate this, we describe our most recent methods for an Atlantic sea scallop (Placopecten magellanicus) drop camera survey to provide a procedural example and representative results. The procedure is divided into three critical steps that include survey design, data collection, and data products. The influence of scallop behavior and the survey goal of providing an independent assessment of the U.S. sea scallop resource on the survey procedure are then discussed in the context of generalizing the method. Overall, the broad applicability and flexibility of the University of Massachusetts Dartmouth School for Marine Science and Technology (SMAST) drop camera survey demonstrates the method could be generalized and applied to a variety of sessile invertebrates or habitat focused research.

Image based surveying is an increasingly practical, non-invasive method to sample the marine environment. We present the protocol of a drop camera survey that estimates the abundance and distribution of the Atlantic sea scallop (Placopecten magellanicus). We discuss how this protocol can be generalized for application to other benthic macroinvertebrates.

Underwater imaging has long been used in the field of marine ecology but decreasing costs of high-resolution cameras and data storage have made the ...

The Power of Simplicity: Sea Urchin Embryos as in Vivo Developmental Models for Studying Complex Cell-to-cell Signaling Network Interactions

Remarkably few cell-to-cell signal transduction pathways are necessary during embryonic development to generate the large variety of cell types and tissues in the adult body form. Yet, each year more components of individual signaling pathways are discovered, and studies indicate that depending on the context there is significant cross-talk among most of these pathways. This complexity makes studying cell-to-cell signaling in any in vivo developmental model system a difficult task. In addition, efficient functional analyses are required to characterize molecules associated with signaling pathways identified from the large data sets generated by next generation differential screens. Here, we illustrate a straightforward method to efficiently identify components of signal transduction pathways governing cell fate and axis specification in sea urchin embryos. The genomic and morphological simplicity of embryos similar to those of the sea urchin make them powerful in vivo developmental models for understanding complex signaling interactions. The methodology described here can be used as a template for identifying novel signal transduction molecules in individual pathways as well as the interactions among the molecules in the various pathways in many other organisms.

The Power of Simplicity: Sea Urchin Embryos as in Vivo Developmental Models for Studying Complex Cell-to-cell Signaling Network Interactions

This video article details a straightforward in vivo methodology that can be used to systematically and efficiently characterize components of complex signaling pathways and regulatory networks in many invertebrate embryos.

Remarkably few cell-to-cell signal transduction pathways are necessary during embryonic development to generate the large variety of cell types and ...

Design and Use of an Apparatus for Quantifying Bivalve Suspension Feeding at Sea

As shellfish aquaculture moves from coastal embayments and estuaries to offshore locations, the need to quantify ecosystem interactions of farmed bivalves (i.e., mussels, oysters, and clams) presents new challenges. Quantitative data on the feeding behavior of suspension-feeding mollusks is necessary to determine important ecosystem interactions of offshore shellfish farms, including their carrying capacity, the competition with the zooplankton community, the availability of trophic resources at different depths, and the deposition to the benthos. The biodeposition method is used to quantify feeding variables in suspension-feeding bivalves in a natural setting and represents a more realistic proxy than laboratory experiments. This method, however, relies upon a stable platform to satisfy the requirements that water flow rates supplied to the shellfish remain constant and the bivalves are undisturbed. A flow-through device and process for using the biodeposition method to quantify the feeding of bivalve mollusks were modified from a land-based format for shipboard use by building a two-dimensional gimbal table around the device. Planimeter data reveal a minimal pitch and yaw of the chambers containing the test shellfish despite boat motion, the flow rates within the chambers remain constant, and operators are able to collect the biodeposits (feces and pseudofeces) with sufficient consistency to obtain accurate measurements of the bivalve clearance, filtration, selection, ingestion, rejection, and absorption at offshore shellfish aquaculture sites.

A flow-through device for using the biodeposition method to quantify filtration and feeding behavior of bivalve mollusks was modified for shipboard use. A two-dimensional gimbal table built around the device isolates the apparatus from boat motion, thereby allowing the accurate quantification of bivalve filtration variables at offshore shellfish aquaculture sites.

As shellfish aquaculture moves from coastal embayments and estuaries to offshore locations, the need to quantify ecosystem interactions of farmed bivalves ...

A Robotic Platform to Study the Foreflipper of the California Sea Lion

The California sea lion (Zalophus californianus), is an agile and powerful swimmer. Unlike many successful swimmers (dolphins, tuna), they generate most of their thrust with their large foreflippers. This protocol describes a robotic platform designed to study the hydrodynamic performance of the swimming California sea lion (Zalophus californianus). The robot is a model of the animal&#39;s foreflipper that is actuated by motors to replicate the motion of its propulsive stroke (the &#39;clap&#39;). The kinematics of the sea lion&#39;s propulsive stroke are extracted from video data of unmarked, non-research sea lions at the Smithsonian Zoological Park (SNZ). Those data form the basis of the actuation motion of the robotic flipper presented here. The geometry of the robotic flipper is based a on high-resolution laser scan of a foreflipper of an adult female sea lion, scaled to about 60% of the full-scale flipper. The articulated model has three joints, mimicking the elbow, wrist and knuckle joint of the sea lion foreflipper. The robotic platform matches dynamics properties&#8212;Reynolds number and tip speed&#8212;of the animal when accelerating from rest. The robotic flipper can be used to determine the performance (forces and torques) and resulting flowfields.

A robotic platform is described that will be used to study the hydrodynamic performance&#8212;forces and flowfields&#8212;of the swimming California sea lion. The robot is a model of the animal&#39;s foreflipper that is actuated by motors to replicate the motion of its propulsive stroke (the &#39;clap&#39;).

The California sea lion (Zalophus californianus), is an agile and powerful swimmer. Unlike many successful swimmers (dolphins, tuna), they generate most ...

Engineering

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 ...

Assessing Intertidal Populations of the Invasive European Green Crab

Invasive species have caused major disruptions to ecosystems worldwide. The European green crab invaded North America in the 1800s and is considered one of the world&#8217;s 100 worst invaders by the IUCN. Observations of spatiotemporal green crab population dynamics are essential for predicting and managing the ecological and economic impacts of this harmful invasive species. This protocol was developed in an effort to create a standardized method for assessing green crab population dynamics in the rocky intertidal zone of New England and Atlantic Canada. The protocol was designed to be accessible to multiple users including researchers, educators, students, and citizen scientists. Although it was designed for surveying crab populations, this protocol is easy to adapt and could be used for any number of intertidal species. The resulting data collected using this protocol has a wide range of uses, including to inform ecological research, conservation efforts, mitigation strategies, and fishery development, as well as for educational outreach purposes.

Understanding spatiotemporal patterns in green crab population dynamics is essential for predicting and managing the ecological and economic impacts of this harmful invasive species. This protocol was developed in an effort to create a standardized method for assessing green crab populations in the rocky intertidal zone of the Northwest Atlantic.

Invasive species have caused major disruptions to ecosystems worldwide. The European green crab invaded North America in the 1800s and is considered one ...

A Protocol for Bioinspired Design: A Ground Sampler Based on Sea Urchin Jaws

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