Name: establishing an octopus ecosystem for biomedical and bioengineering research
Uploaded: 2021-09-22T15:00:00
Description: Watch this Scientific Journal Video about Establishing an Octopus Ecosystem for Biomedical and Bioengineering Research at JoVE.com

This work was supported by NIH UF1NS115817 (G.P.). G.P. is partially supported by NIH grants R01NS072171 and R01NS098231. We would like to thank Patrick Zakrzewki and Mohammed Farhoud from Emit Imaging for the help and support in collecting and visualizing the data on the Xerra Imaging Platform. MSU has a research agreement with Bruker Biospin.

All animal studies were approved by the Institutional Animal Care and Use Committee (IACUC) of Michigan State University.
1. Octopus tank equipment set-up
<ol>
	<li>First, obtain all non-biological materials for an aquarium that will be incorporated into the marine environmental system, as shown in the Table of Materials. Sizes are provided in inches.</li>
	<li>Wash all tubing, piping, and filter system parts with 70% ethanol and deionized (DI) water prior to the installatio...

System Setup: 
The aquarium ecosystem has been developed in a way that both mechanical and biological methods of filtering and oxygenating the water are employed. The filtering elements of the system utilize sock filters, protein skimmers, and regular cleaning to maintain nitrogen and oxygen levels. More importantly, we also rely on marine microorganisms to consume the dangerous nitrogenous compounds and other biological waste as well as aerate the water through processes of photosynthesis. Addition...

New concepts in biomedical research and biomedical engineering are often inspired by identifying specific strategies that biological species possess to address environmental and physiological conditions and challenges. For example, understanding the fluorescence properties in fireflies has led to the development of new fluorescent sensors that can report cellular activity in other model organisms1; identifying ion channels activated by light in algae has led to the development of cellular and temporal specific light-based-neuromodulation2,3,4,5; discovering proteins in glass catfish that navigate according to the Earth&#39;s magnetic field has led to the development of magnetic-based-neuromodulation6,7,8,9,10,11; understanding the siphon reflex in Aplysia has been instrumental to understanding the cellular basis of behavior12,13,14.
Researchers continue to expand on the current bioengineering and phylogenetic toolbox by taking advantage of the unique strengths and novel perspectives on physiological functions that non-conventional lab species hold. Federal agencies are beginning to support these lines of studies by funding novel work performed on diverse species.
One genus of animals with unique anatomy and regeneration capabilities as well as the adaptive control of each of its arms, fascinating biologists and engineers, and captivating audiences from every part of the society is the Octopus17. Indeed, many aspects of the octopus&#39; physiology and behavior have been studied over the past decades15,16,17,18,19,20,21,22,23,24,25,26. However, recent developments in molecular and evolutionary biology, robotics, motion recording, imaging, machine learning, and electrophysiology accelerate discoveries related to octopus physiology and behavior and translate them to innovative bioengineering strategies27,28,29,30,31,32,33,34,35,36,37,38,39.
Here we describe how to set up and maintain octopus husbandry, which would be of interest and relevance to scientists and engineers from different backgrounds, scientific interests, and goals. Nevertheless, our results focus on the application of octopuses in neuroscience and neuroengineering research. The octopus has a highly developed nervous system with 45 million neurons in the central brain, 180 million neurons in the optic lobes, and additional 350 million neurons in the eight axial cords and peripheral ganglia; by comparison, a dog has a similar number of neurons and a cat only half of it40. Unlike the vertebrate nervous system, there are only 32K efferent and 140K afferent fibers connecting the millions of neurons in the octopus&#39; brain to the millions of neurons in each of their arm&#39;s axial cords40,41,42. These relatively few interconnecting fibers suggest that most of the details for the execution of the motor programs are performed in the axial cord itself, emphasizing the uniquely distributed neuronal control the octopuses possess. The octopus&#39;s arms have extraordinary fine motor control enabling them manipulation skills such as opening jar lids, even when they are inside the container. This highly developed prehensile motor capability is unique to the class of Cephalopods (octopus, cuttlefish and squid)43.
Indeed, through hundreds of millions of years of evolution, the octopus has developed a remarkable and sophisticated genome and physiological system43,44 that has inspired new development and progress across scientific and engineering fields. For example, a water-resistant adhesive patch based on the anatomical structure of the octopus&#39; suckers can stick to wet and dry surfaces45; a synthetic camouflaging material inspired by the&#160;octopus&#39; camouflage&#160;skin can transform a flat, 2D surface to a three-dimensional one with bumps and pits46. Miniature soft and autonomous robots (i.e., Octobots) that in the future could serve as surgical tools inside the body47; and an arm (i.e., OctoArm) attached to a tank-like robot48 have also been developed. Many species of octopuses are used in biomedical research e.g., Octopus vulgaris, Octopus sinensis, Octopus variabilis, and Octopus bimaculoides (O. bimaculoides); the&#160;O. vulgaris and O. bimaculoides being the most common34,49,50. The recent sequencing of different octopus genomes makes this genus of particular interest and opens new frontiers in octopus research34,43,51,52.
O. bimaculoides used in our set-up is a medium-sized species of octopus, first discovered in 1949, that can be found in shallow waters off the Northeast Pacific coast from central California to the South of Baja California peninsula17. It can be recognized by the false eyespots on its mantle below its eyes. Compared to&#160;Giant Pacific Octopus (Enteroctopus dofleini) and Common Octopus (O. vulgaris), the California Two-Spot Octopus (O. bimaculoides)&#160;is relatively small in size, starting out smaller than a few centimeters, growing fast as a juvenile. When raised within a laboratory, the adult mantle size can grow to an average size of 100 cm and weigh up to 800 g53,54. Octopuses have a rapid growth period within their first 200 days; by then, they are considered adults and continue to grow throughout the rest of their life55,56,57. Octopuses can be cannibalistic, especially when&#160;both sexes are housed together within a tank; therefore, they need to be housed individually in separate tanks58.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>1-3/4 in. Drill Bit</td>
			<td>Home Depot</td>
			<td>204074205</td>
			<td>Glass cutting tool 
			Part number:1</td>
		</tr>
		<tr>
			<td>1&#34; flow sensors</td>
			<td>Neptune Systems</td>
			<td>Local Dealer</td>
			<td>Pipe with sensor to measure water flow 
			Part number:2</td>
		</tr>
		<tr>
			<td>1&#34; Slip Bulkhead Strainer</td>
			<td>Bulk Reef Supply</td>
			<td>207113</td>
			<td>Strainer for water leaving tank 
			Part number:3</td>
		</tr>
		<tr>
			<td>10 gallon tank</td>
			<td>Pruss Pets</td>
			<td>Local Dealer</td>
			<td>Fiddler crab holding tank 
			Part number:4</td>
		</tr>
		<tr>
			<td>4 inch X 12 inch 200 Micron Nylon Monofiliment Mesh Filter Sock w/ Plastic Ring</td>
			<td>AQUAMAXX</td>
			<td>UJ41171</td>
			<td>Filter for large organic matter in sump 
			Part number:5</td>
		</tr>
		<tr>
			<td>40 gallon aquarium</td>
			<td>Pruss Pets</td>
			<td>Local Dealer</td>
			<td>4 Food aquarium tanks 
			Part number:6</td>
		</tr>
		<tr>
			<td>60g poly tanks - rectangle</td>
			<td>Pruss Pets</td>
			<td>Local Dealer</td>
			<td>2 Water Storage (salt and freshwater) 
			Part number:7</td>
		</tr>
		<tr>
			<td>Active Aqua 1/10th HP Hydroponic or Aquarium Chiller 2018 Model</td>
			<td>WayWe</td>
			<td>719574198463</td>
			<td>For cooling water continuously 
			Part number:8</td>
		</tr>
		<tr>
			<td>ALAZCO 2 Soft-Grip Handle Heavy-Duty Tile Grout Brush</td>
			<td>ALAZCO</td>
			<td>B06W2FT5V5</td>
			<td>Tank Cleaning 
			Part number:9</td>
		</tr>
		<tr>
			<td>Ammonia Testing Kit</td>
			<td>Aquarium Pharmaceuticals</td>
			<td>33D</td>
			<td>For water testing 
			Part number:10</td>
		</tr>
		<tr>
			<td>Apex system WiFi</td>
			<td>Neptune Systems</td>
			<td>Local Dealer</td>
			<td>System connection for off site monitoring 
			Part number:11</td>
		</tr>
		<tr>
			<td>API Aquarium Test Kit</td>
			<td>Amazon</td>
			<td>B001EUE808</td>
			<td>For water testing 
			Part number:12</td>
		</tr>
		<tr>
			<td>API Copper Test Kit</td>
			<td>Amazon</td>
			<td>B0006JDWH8</td>
			<td>For water testing 
			Part number:13</td>
		</tr>
		<tr>
			<td>Aqua Ultraviolet Classic UV 25 Watt Series Units</td>
			<td>Aqua Ultraviolet</td>
			<td>A00028</td>
			<td>For removing bacteria leaving sump system 
			Part number:14</td>
		</tr>
		<tr>
			<td>AquaClear 50 Foam Filter Inserts, 3 pack</td>
			<td>Aquaclear</td>
			<td>A1394</td>
			<td>Food Tank Carbon Filter Inserts 
			Part number:15</td>
		</tr>
		<tr>
			<td>Aqueon QuietFlow LED PRO Aquarium Power Filter 30</td>
			<td>Aqueon</td>
			<td>100106082</td>
			<td>Food tank filtering units 
			Part number:16</td>
		</tr>
		<tr>
			<td>Auto Top Off Kit (ATK) (Each includes 1 FMM module, 2 optical sensors and 1 float)</td>
			<td>Neptune Systems</td>
			<td>Local Dealer</td>
			<td>For freshwater tank 
			Part number:17</td>
		</tr>
		<tr>
			<td>Automatic top off from RODI (LLC)</td>
			<td>Neptune Systems</td>
			<td>Local Dealer</td>
			<td>From water storage to octopus tanks 
			Part number:18</td>
		</tr>
		<tr>
			<td>Banded Trochus Snail</td>
			<td>LiveAquaria</td>
			<td>CN-112080</td>
			<td>For algae bin 
			Part number:19</td>
		</tr>
		<tr>
			<td>Chaetomorpha Algae, Aquacultured</td>
			<td>LiveAquaria</td>
			<td>BVJ-76354</td>
			<td>For algae bin 
			Part number:20</td>
		</tr>
		<tr>
			<td>Clams - Live, Hard Shell, Cherrystone, Wild, USA Dozen</td>
			<td>Fulton Fish Market</td>
			<td>N/A</td>
			<td>Live food 
			Part number:21</td>
		</tr>
		<tr>
			<td>Classic Sea Salt Mix - Tropic Marin</td>
			<td>Bulk Reef Supply</td>
			<td>211813</td>
			<td>Salt for tank water 
			Part number:22</td>
		</tr>
		<tr>
			<td>Clear Masterkleer Soft PVC Plastic Tubing, for Air and Water, 3/4&#34; ID,&#160;1&#34; OD</td>
			<td>McMaster</td>
			<td>5233K71</td>
			<td>Cleaning tool 
			Part number:23</td>
		</tr>
		<tr>
			<td>Continuum Aquablade-P Acrylic Safe Algae Scraper W/ Plastic Blade - 15 Inch</td>
			<td>Marine Depot</td>
			<td>4C31001</td>
			<td>Cleaning tool 
			Part number:24</td>
		</tr>
		<tr>
			<td>Copper Testing Kit</td>
			<td>Aquarium Pharmaceuticals</td>
			<td>65L</td>
			<td>For water testing 
			Part number:25</td>
		</tr>
		<tr>
			<td>Curve Refugium CREE LED Aquarium Light</td>
			<td>Eshopps</td>
			<td>6500K</td>
			<td>Algae bin light 
			Part number:26</td>
		</tr>
		<tr>
			<td>Eheim 1262 return pumps</td>
			<td>EHEIM</td>
			<td>1250219</td>
			<td>Pump for storage tanks 
			Part number:27</td>
		</tr>
		<tr>
			<td>Eshopps R-100 Refugium Sump GEN 3</td>
			<td>Eshopps</td>
			<td>15000</td>
			<td>Sump system 
			Part number:28</td>
		</tr>
		<tr>
			<td>Ethyl Alcohol, 200 Proof</td>
			<td>Sigma-Aldrich</td>
			<td>64-17-5</td>
			<td>Anesthesia 
			Part number:29</td>
		</tr>
		<tr>
			<td>Extech DO600 ExStik II Dissolved Oxygen Meter</td>
			<td>Extech</td>
			<td>DO600</td>
			<td>Oxygen measurment 
			Part number:30</td>
		</tr>
		<tr>
			<td>Fiddler Crabs; live; dozen</td>
			<td>NORTHEAST BRINE SHRIMP</td>
			<td>N/A</td>
			<td>Live food 
			Part number:31</td>
		</tr>
		<tr>
			<td>Filter Cartrages</td>
			<td>Aqueon</td>
			<td>100106087</td>
			<td>Food tank filters 
			Part number:32</td>
		</tr>
		<tr>
			<td>Florida Crushed Coral Dry Sand - CaribSea</td>
			<td>Bulk Reef Supply</td>
			<td>212959</td>
			<td>Sedimate for bottom of tank 
			Part number:33</td>
		</tr>
		<tr>
			<td>FMM module</td>
			<td>Neptune Systems</td>
			<td>Local Dealer</td>
			<td>Controller for apex system 
			Part number:34</td>
		</tr>
		<tr>
			<td>Fritz-Zyme TurboStart 900 - Fritz</td>
			<td>Bulk Reef Supply</td>
			<td>213036</td>
			<td>Bacteria start 
			Part number:35</td>
		</tr>
		<tr>
			<td>Hand Operated Drum Pump, Siphon, Basic Pump with Spout, For Container Type Bucket, Pail</td>
			<td>Granger</td>
			<td>38Y789</td>
			<td>Water Hand Pump 
			Part number:36</td>
		</tr>
		<tr>
			<td>High pH Testing Kit</td>
			<td>Aquarium Pharmaceuticals</td>
			<td>27</td>
			<td>For water testing 
			Part number:37</td>
		</tr>
		<tr>
			<td>Imagitarium Fine Mesh Net for Shrimp</td>
			<td>Petco</td>
			<td>2580993</td>
			<td>Shrimp and fish transfer net 
			Part number:38</td>
		</tr>
		<tr>
			<td>Leak Detection Kit (LDK) - Includes FMM module plus 2 ALD sensors</td>
			<td>Neptune Systems</td>
			<td>Local Dealer</td>
			<td>Placed on floor to detect water 
			Part number:39</td>
		</tr>
		<tr>
			<td>Lee`S Algae Scrubber Pad Jumbo - Glass</td>
			<td>Marine Depot</td>
			<td>LE12007</td>
			<td>Cleaning tool 
			Part number:40</td>
		</tr>
		<tr>
			<td>Live rocks</td>
			<td>Pruss Pets</td>
			<td>Local Dealer</td>
			<td>Habitat for octopus 
			Part number:41</td>
		</tr>
		<tr>
			<td>Long Bottle Cleaning Brush 17&#34; Extra Long</td>
			<td>Haomaomao</td>
			<td>B07FS7J7PN</td>
			<td>Tank Cleaning 
			Part number:42</td>
		</tr>
		<tr>
			<td>Magnesium chloride</td>
			<td>Sigma-Aldrich</td>
			<td>M1028-100ML</td>
			<td>Euthanasia 
			Part number:43</td>
		</tr>
		<tr>
			<td>Magnetic Probe Rack</td>
			<td>Neptune Systems</td>
			<td>Local Dealer</td>
			<td>For holding apex sensor probes 
			Part number:44</td>
		</tr>
		<tr>
			<td>Marine Ghost Shrimp</td>
			<td>NORTHEAST BRINE SHRIMP</td>
			<td>N/A</td>
			<td>Live food 
			Part number:45</td>
		</tr>
		<tr>
			<td>Marineland C-Series Canister Carbon Bags Filter Media, 2 count</td>
			<td>Chewy</td>
			<td>98331</td>
			<td>For elevated copper levels 
			Part number:46</td>
		</tr>
		<tr>
			<td>Nitra-Zorb Bag</td>
			<td>Aquarium Pharmaceuticals</td>
			<td>AP2213</td>
			<td>Absorbes nirtogen compounds 
			Part number:47</td>
		</tr>
		<tr>
			<td>Nitrate Testing Kit</td>
			<td>Aquarium Pharmaceuticals</td>
			<td>LR1800</td>
			<td>For water testing 
			Part number:48</td>
		</tr>
		<tr>
			<td>Nitrite Testing Kit</td>
			<td>Aquarium Pharmaceuticals</td>
			<td>26</td>
			<td>For water testing 
			Part number:49</td>
		</tr>
		<tr>
			<td>Pawfly 2 Inch Air Stones Cylinder 6 PCS Bubble Diffuser Airstones for Aquarium Fish Tank Pump Blue</td>
			<td>Amazon</td>
			<td>B076S56XWX</td>
			<td>Aierating water 
			Part number:50</td>
		</tr>
		<tr>
			<td>Penn Plax Airline Tubing for Aquariums &#8211;Clear and Flexible Resists Kinking, 8 Feet Standard</td>
			<td>Amazon</td>
			<td>B0002563MM</td>
			<td>Tubing for connecting air pump to air stone 
			Part number:51</td>
		</tr>
		<tr>
			<td>Plumbing with unions/valves plus 3/4&#34; flex hose</td>
			<td>Pruss Pets</td>
			<td>Local Dealer</td>
			<td>Water transport 
			Part number:52</td>
		</tr>
		<tr>
			<td>PM1 module</td>
			<td>Neptune Systems</td>
			<td>Local Dealer</td>
			<td>Power control module for apex 
			Part number:53</td>
		</tr>
		<tr>
			<td>Protein skimmer</td>
			<td>Reef Octopus</td>
			<td>AC20284</td>
			<td>Removes biowaste from system 
			Part number:54</td>
		</tr>
		<tr>
			<td>PVC Apex Mounting board, grommets, wire mounts</td>
			<td>Neptune Systems</td>
			<td>Local Dealer</td>
			<td>Helps ensure organization for wires and tubing within system 
			Part number:55</td>
		</tr>
		<tr>
			<td>PVC Regular Cement and 4-Ounce NSF Purple Primer</td>
			<td>Amazon</td>
			<td>Oatey - 30246</td>
			<td>For connecting PVC pipes 
			Part number:56</td>
		</tr>
		<tr>
			<td>RODI unit</td>
			<td>Neptune Systems</td>
			<td>Local Dealer</td>
			<td>RO Water 
			Part number:57</td>
		</tr>
		<tr>
			<td>Salinity Probes</td>
			<td>HANNA probes</td>
			<td>HI98319</td>
			<td>Measures salinity of water 
			Part number:58</td>
		</tr>
		<tr>
			<td>Seachem Pristine Aquarium Treatment</td>
			<td>Sea Chem</td>
			<td>1438</td>
			<td>Provides bacteria that break down excess food, waste and detritus 
			Part number:59</td>
		</tr>
		<tr>
			<td>Seachem Stability Fish Tank Stabilizer</td>
			<td>Sea Chem</td>
			<td>116012607</td>
			<td>Seachem Stability will rapidly and safely establish the aquarium biofilter in freshwater and marine systems 
			Part number:60</td>
		</tr>
		<tr>
			<td>Set of lexan tops</td>
			<td>Pruss Pets</td>
			<td>Local Dealer</td>
			<td>Aquarium tank lids 
			Part number:61</td>
		</tr>
		<tr>
			<td>Set of Various extended length aquabus cables</td>
			<td>Neptune Systems</td>
			<td>Local Dealer</td>
			<td>Cables for Apex system 
			Part number:62</td>
		</tr>
		<tr>
			<td>SLSON Aquarium Algae Scraper Double Sided Sponge Brush Cleaner Long Handle Fish Tank Scrubber for Glass Aquariums</td>
			<td>Amazon</td>
			<td>B07DC2TZCJ</td>
			<td>Cleaning tool 
			Part number:63</td>
		</tr>
		<tr>
			<td>Standard-Wall PVC Pipe Fitting for Water, 45 Degree Elbow Adapter, 3/4 Socket Female x 3/4 Socket Male</td>
			<td>McMaster</td>
			<td>4880K189</td>
			<td>PVC pipe 
			Part number:64</td>
		</tr>
		<tr>
			<td>Standard-Wall&#160;PVC Pipe Fitting for Water, 90 Degree&#160;Elbow&#160;Adapter,&#160;1 Socket Female x 1 Socket Male</td>
			<td>McMaster</td>
			<td>4880K773</td>
			<td>PVC pipe 
			Part number:65</td>
		</tr>
		<tr>
			<td>Standard-Wall&#160;PVC Pipe Fitting for Water, Adapter,&#160;1&#160;Socket-Connect&#160;Female x 1 Barbed Male</td>
			<td>McMaster</td>
			<td>4880K415</td>
			<td>PVC pipe 
			Part number:66</td>
		</tr>
		<tr>
			<td>Standard-Wall&#160;PVC Pipe Fitting for Water, Straight&#160;Reducer,&#160;2 Socket Female x&#160;3/4&#160;Socket Female</td>
			<td>McMaster</td>
			<td>4880K008</td>
			<td>PVC pipe 
			Part number:67</td>
		</tr>
		<tr>
			<td>Standard-Wall&#160;PVC Pipe Fitting for Water, Tee Connector,&#160;White,&#160;1 Size&#160;Socket-Connect&#160;Female</td>
			<td>McMaster</td>
			<td>4880K43</td>
			<td>PVC pipe 
			Part number:68</td>
		</tr>
		<tr>
			<td>Standard-Wall&#160;Unthreaded Rigid PVC Pipe for Water, 1 Pipe Size, 10 Feet Long</td>
			<td>McMaster</td>
			<td>48925K13</td>
			<td>PVC pipe 
			Part number:69</td>
		</tr>
		<tr>
			<td>Standard-Wall Unthreaded Rigid PVC Pipe for Water, 3/4 Pipe Size, 5 Feet Long</td>
			<td>McMaster</td>
			<td>48925K92</td>
			<td>PVC pipe 
			Part number:70</td>
		</tr>
		<tr>
			<td>Structural FRP Fiberglass Sheet, 48&#34; Wide x 96&#34; Long, 1/4&#34; Thick</td>
			<td>McMaster</td>
			<td>8537K15</td>
			<td>Table top material 
			Part number:71</td>
		</tr>
		<tr>
			<td>Structural FRP Fiberglass Square Tube, 10 Feet Long, 2&#34; Wide x 2&#34; High Outside, 1/8&#34; Wall Thickness</td>
			<td>McMaster</td>
			<td>8548K33</td>
			<td>Structural table materal 
			Part number:72</td>
		</tr>
		<tr>
			<td>Tank Sediment</td>
			<td>TopDawg Pet Supply</td>
			<td>8479001207</td>
			<td>Sediment for bottom of fiddler crab tank 
			Part number:73</td>
		</tr>
		<tr>
			<td>Temperature probe</td>
			<td>Neptune Systems</td>
			<td>Local Dealer</td>
			<td>Temperature probe for tanks 
			Part number:74</td>
		</tr>
		<tr>
			<td>Tetra TetraMarine Large Saltwater Flakes for all Marine Fish</td>
			<td>Amazon</td>
			<td>B00025K0US</td>
			<td>Fish, shrimp, and crab food 
			Part number:75</td>
		</tr>
		<tr>
			<td>Tetra Whisper Aquarium Air Pump for 10 gallon Aquariums</td>
			<td>Petco</td>
			<td>2335234</td>
			<td>Air pump for smaller tanks 
			Part number:76</td>
		</tr>
		<tr>
			<td>Thick-Wall Through-Wall Pipe Fitting, for Water, PVC Connector, 1 Socket-Connect Female</td>
			<td>McMaster</td>
			<td>36895K843</td>
			<td>PVC pipe 
			Part number:77</td>
		</tr>
		<tr>
			<td>Vectra s2 pump</td>
			<td>Bulk Reef Supply</td>
			<td>212141</td>
			<td>Aquarium Pump 
			Part number:78</td>
		</tr>
		<tr>
			<td>Water Pump</td>
			<td>TACKLIFE</td>
			<td>GHWP1A</td>
			<td>Pump for cleaning tanks 
			Part number:79</td>
		</tr>
		<tr>
			<td>Wyze Cam v2 1080p HD Indoor WiFi Smart Home Camera with Night Vision</td>
			<td>Amazon</td>
			<td>B076H3SRXG</td>
			<td>DeepLabCut Recording 
			Part number:80</td>
		</tr>
	</tbody>
</table>

establishing an octopus ecosystem for biomedical and bioengineering research

Many developments in biomedical research have been inspired by discovering anatomical and cellular mechanisms that support specific functions in different species. The octopus is one of these exceptional animals that has given scientists new insights into the fields of neuroscience, robotics, regenerative medicine, and prosthetics. Research with this species of cephalopods requires the set-up of complex facilities and intensive care for both the octopus and its ecosystem that is critical for the project&#39;s success. This system requires multiple mechanical and biological filtering systems to provide a safe and clean environment for the animal. Along with the control system, specialized routine maintenance and cleaning are required to effectively keep the facility operating long term. It is advised to provide an enriched environment to these intelligent animals by changing the tank&#39;s landscape, incorporating a variety of prey, and introducing challenging tasks for them to work through. Our results include MRI and a whole-body autofluorescence imaging as well as behavioral studies to better understand their nervous system. Octopuses possess unique physiology that can impact many areas of biomedical research. Providing them with a sustainable ecosystem is the first crucial step in uncovering their distinct capabilities.

All the animals in our studies were obtained from the wild, and thus their exact age could not be determined and their stay in the lab was variable. Octopus condition was observed daily. We did not see parasites, bacteria, skin damage, or abnormal behavior. The average weight of animals was 170.38 +/- 77.25 g. Each animal inhabited their own 40-gallon tank. The mean &#177; standard deviation for the parameters recorded for a tank over a week were: pH 8.4 &#177; 0.0, salinity 34.06 &#177; 0.61 ppt, temperature 18.7 &#177;...

Watch this Scientific Journal Video about Establishing an Octopus Ecosystem for Biomedical and Bioengineering Research at JoVE.com

Establishing an Octopus Ecosystem for Biomedical and Bioengineering Research

Understanding the unique physiological and anatomical structures of octopuses can greatly impact biomedical research. This guide demonstrates how to set-up and maintain a marine environment to accommodate this species and includes state-of-the-art imaging and analytical approaches to visualize octopus' nervous system anatomy and function.

Many developments in biomedical research have been inspired by discovering anatomical and cellular mechanisms that support specific functions in different ...

Our protocol is significant because it guides researchers on maintaining an octopus environment, explaining how to set up the aquarium, daily care of octopuses. This technique guides new researchers without octopus care experiences set up an aquarium environment for octopuses in a laboratory and maintaining healthy octopuses for research. It is important for new researchers with octopuses to realize that these animals require strong commitment.They require daily care and the water quality needs to be continuously monitored in a stable condition before the animals even arrive. One week after the octopus tanks are matured, set up a separate shrimp tank by transferring eight gallons of matured saltwater to the tank, then add 15 kilograms of crushed coral to the bottom of the tank. Also add a few live rocks to provide hiding spots for molting.Next, attach a canister filter to the edge of the tank and set it up as directed by the manufacturer. Add an air pump next to the tank connected to a tube with an attached airstone placed into the tank. Once the crushed coral sediment dissipates the shrimp can be added to the tank, To add the shrimp on arrival, first move the shrimp from the shipping water to the small intermediatory saltwater tank for five minutes to remove bio waste, then the shrimp can be added directly to the tank.Mosquito fish, on arrival, can be added directly to the shrimp tank. Feed the shrimp and fish with fish flakes, dead vegetation, or algae. Every week, clean the filter, change the filter pads, and change 25%of the water.Using water testing kits, check nitrogen, pH, and temperature parameters daily in the food tanks. If water nitrogen parameters remain high, change the water more frequently and add a nitrogen absorbent bag. If problems persist longer than a month, move the shrimp to a larger tank.For the crab tank, pile 10 kilograms of pebbles on one side to create dry land, and fill the other side with one gallon of saltwater. Then, add fiddler crabs directly into the tank. Crabs will spend most of their lives on land, but can be underwater for a few days at a time, making a partially-underwater tank crucial for their long term survival.Feed the fiddler crabs once per day by adding fish flakes into the dish on the area of the tank. Clean the tanks weekly by first removing the crabs and then changing 100%of the salt water and cleaning the pebbles. Place marine bivalve molluscs, such as clams and mussels, within the octopus saltwater tank.These molluscs will open up, providing another water filtering mechanism. Before introducing the octopus into the tank, ensure that the ammonia, nitrite, and nitrate levels are below 5, 25, and 10 parts per million, respectively. Have a water hand pump available to remove octopus ink from the tank.Two people are required for this procedure. On arrival, place the bag on the scale and record the weight of the bag with the octopus. Add an airstone to the bag to increase the water oxygenation while transferring the animal to their tank.Measure the shipping water's temperature and salinity and record cases of prolonged illness after shipment. Without transferring any water from the bag to the tank, hang the transport bag over the corner of the tank with the bag partially-submerged in the tank water to begin changing the temperature of the transportation bag. Remove 10%of the water from the bag and dump it down the sink, then add the same amount of water from the tank to the bag.Repeat every 10 minutes until the water temperature in the bag is no more than one degree different than the water temperature in the tank. Then, wear gloves and place both hands under the octopus to provide support during the transfer. The second person will need to gently pull the suctioned arms from the side of the bag.Once the octopus is out of the bag, move it quickly into the water of its new habitat, transferring as little water from the shipping bag as possible. Use the hand pump to remove any ink the octopus releases when in the tank. Now weigh the bag with water to obtain the approximate weight of the animal.For the first two weeks after arrival, monitor the octopus's daily consumption, which should be around 4 to 8%of its weight. The octopus should be checked four times a day. This can be decreased to twice per day after two weeks.Weigh the animal every two weeks and adjust food consumption as needed. Use a commercially-available saltwater testing kit for pH, ammonia, nitrite, and nitrate levels. Add the kit-directed amount of tank water to the four test tubes provided with the kit.As specified on the testing kit, add the amount of calorimetric reactant to the corresponding tube. If the ammonia, nitrite, and nitrate levels are above 5, 25, and 10 parts per million, respectively, wash the biomass out of the sock filter, or change to a new sock filter. Additionally, clean out biomass from the top of the skimmer with a brush and add additional denitrifying bacteria to the tank.If problems persist, then replace 25%of the water with fresh saltwater. Using a hand pump, remove all dead crab and shrimp carcasses from the tank as well as any octopus fecal matter. Remove all the remaining living crabs from the tank and move them back to the storage tank.Next, rearrange large objects within the tank. Then, introduce half the number of crabs that the octopus would eat daily to the tank. Feed defrosted shrimp or small male fiddler crabs to juvenile octopuses.Depending on the experiment, crabs and shrimp can be introduced anywhere in the tank, or to the octopus directly. Offer five ghost shrimp daily. To provide a variety of food to the octopus, give one live clam or mussel once a week, and always maintain three mosquito fish inside the tank.For weekly sanitation, shut down the skimmer, pump, and algae bin lights before cleaning the pump system, then turn off the automatic valve of the system before removing water. Finally, remove the skimmer and all the water only from the sump system. Lightly scrub the algae bin to remove most of the biomass from its walls and clean the rest of the sump area with a brush, then remove the sock filter, clean it out with vinegar, and let it dry.Rotate with another sock filter each week, replacing with new ones every three months. After cleaning out the biomass from the top of the skimmer, put the skimmer back into the system and begin refilling with salt water. When the pump area begins to fill, turn all the systems back on.Stop adding water when the automatic top of the float valve is in the off position. Unscrewing test tubes is a valuable test for understanding the sensorimotor function, as well as learning the memory capabilities of octopuses. This test was performed daily on three octopuses, and it took the octopuses four days on average to learn how to open the test tube.Shown here are ultra-high spatial resolution MRI images of the octopus's brain and arms that, together, form a nervous system containing over 500 million neurons. This is a coronal view of the head, showing the brain and the optic lobes. In the coronal view of the arms, The axial cord is visible in each of the seven arms, and a sagittal view of the suckers demonstrates a complex peripheral nervous structure.Cryo-fluorescence tomography imaging, which generates a three-dimensional morphological image of the entire animal, allowed visualization of the brain and the suckers positioned along the arm at the 470-nanometer wavelength. The digestive system is visualized in the 555-and 640-nanometer wavelengths. After this technique was developed, it was possible to use machine learning tools for tracking and classifying the octopus movements.

establishing-an-octopus-ecosystem-for-biomedical-bioengineering

Research

JoVE Journal

Bioengineering

Graphene Coatings for Biomedical Implants

Atomically smooth graphene as a surface coating has potential to improve implant properties. This demonstrates a method for coating nitinol alloys with nanometer thick layers of graphene for applications as a stent material. Graphene was grown on copper substrates via chemical vapor deposition and then transferred onto nitinol substrates. In order to understand how the graphene coating could change biological response, cell viability of rat aortic endothelial cells and rat aortic smooth muscle cells was investigated. Moreover, the effect of graphene-coatings on cell adhesion and morphology was examined with fluorescent confocal microscopy. Cells were stained for actin and nuclei, and there were noticeable differences between pristine nitinol samples compared to graphene-coated samples. Total actin expression from rat aortic smooth muscle cells was found using western blot. Protein adsorption characteristics, an indicator for potential thrombogenicity, were determined for serum albumin and fibrinogen with gel electrophoresis. Moreover, the transfer of charge from fibrinogen to substrate was deduced using Raman spectroscopy. It was found that graphene coating on nitinol substrates met the functional requirements for a stent material and improved the biological response compared to uncoated nitinol. Thus, graphene-coated nitinol is a viable candidate for a stent material.

Graphene offers potential as a coating material for biomedical implants. In this study we demonstrate a method for coating nitinol alloys with nanometer thick layers of graphene and determine how graphene may influence implant response.

Atomically smooth graphene as a surface coating has potential to improve implant properties. This demonstrates a method for coating nitinol alloys with ...

Establishing Fungal Entomopathogens as Endophytes: Towards Endophytic Biological Control

Beauveria bassiana is a fungal entomopathogen with the ability to colonize plants endophytically. As an endophyte, B. bassiana may play a role in protecting plants from herbivory and disease. This protocol demonstrates two inoculation methods to establish B. bassiana endophytically in the common bean (Phaseolus vulgaris), in preparation for subsequent evaluations of endophytic biological control. Plants are grown from surface-sterilized seeds for two weeks before receiving a B. bassiana treatment of 108 conidia/ml (or water) applied either as a foliar spray or a soil drench. Two weeks later, the plants are harvested and their leaves, stems and roots are sampled to evaluate endophytic fungal colonization. For this, samples are individually surface sterilized, cut into multiple sections, and incubated in potato dextrose agar media for 20 days. The media is inspected every 2-3 days to observe fungal growth associated with plant sections and record the occurrence of B. bassiana to estimate the extent of its endophytic colonization. Analyses of inoculation success compare the occurrence of B. bassiana within a given plant part (i.e. leaves, stems or roots) across treatments and controls. In addition to the inoculation method, the specific outcome of the experiment may depend on the target crop species or variety, the fungal entomopathogen species strain or isolate used, and the plant's growing conditions.

This protocol demonstrates two inoculation methods to introduce the fungal entomopathogen Beauveria bassiana as an endophyte in the common bean (Phaseolus vulgaris), in preparation for subsequent evaluations of endophytic biological control.

 Beauveria bassiana is a fungal entomopathogen with the ability to colonize plants endophytically. As an endophyte, B. bassiana may play a role in ...

Designing Silk-silk Protein Alloy Materials for Biomedical Applications

Fibrous proteins display different sequences and structures that have been used for various applications in biomedical fields such as biosensors, nanomedicine, tissue regeneration, and drug delivery. Designing materials based on the molecular-scale interactions between these proteins will help generate new multifunctional protein alloy biomaterials with tunable properties. Such alloy material systems also provide advantages in comparison to traditional synthetic polymers due to the materials biodegradability, biocompatibility, and tenability in the body. This article used the protein blends of wild tussah silk (Antheraea pernyi) and domestic mulberry silk (Bombyx mori) as an example to provide useful protocols regarding these topics, including how to predict protein-protein interactions by computational methods, how to produce protein alloy solutions, how to verify alloy systems by thermal analysis, and how to fabricate variable alloy materials including optical materials with diffraction gratings, electric materials with circuits coatings, and pharmaceutical materials for drug release and delivery. These methods can provide important information for designing the next generation multifunctional biomaterials based on different protein alloys.

Blending is an efficient approach to generate biomaterials with a broad range of properties and combined features. By predicting the molecular interactions between different natural silk proteins, new silk-silk protein alloy platforms with tunable mechanical resiliency, electrical response, optical transparency, chemical processability, biodegradability, or thermal stability can be designed.

Fibrous proteins display different sequences and structures that have been used for various applications in biomedical fields such as biosensors, ...

Fabricating Superhydrophobic Polymeric Materials for Biomedical Applications

Superhydrophobic materials, with surfaces possessing permanent or metastable non-wetted states, are of interest for a number of biomedical and industrial applications. Here we describe how electrospinning or electrospraying a polymer mixture containing a biodegradable, biocompatible aliphatic polyester (e.g., polycaprolactone and poly(lactide-co-glycolide)), as the major component, doped with a hydrophobic copolymer composed of the polyester and a stearate-modified poly(glycerol carbonate) affords a superhydrophobic biomaterial. The fabrication techniques of electrospinning or electrospraying provide the enhanced surface roughness and porosity on and within the fibers or the particles, respectively. The use of a low surface energy copolymer dopant that blends with the polyester and can be stably electrospun or electrosprayed affords these superhydrophobic materials. Important parameters such as fiber size, copolymer dopant composition and/or concentration, and their effects on wettability are discussed. This combination of polymer chemistry and process engineering affords a versatile approach to develop application-specific materials using scalable techniques, which are likely generalizable to a wider class of polymers for a variety of applications.

Two- and three-dimensional superhydrophobic polymeric materials are prepared by electrospinning or electrospraying biodegradable polymers blended with a lower surface energy polymer of similar composition.

Superhydrophobic materials, with surfaces possessing permanent or metastable non-wetted states, are of interest for a number of biomedical and industrial ...

Fabrication of Mechanically Tunable and Bioactive Metal Scaffolds for Biomedical Applications

Biometal systems have been widely used for biomedical applications, in particular, as load-bearing materials. However, major challenges are high stiffness and low bioactivity of metals. In this study, we have developed a new method towards fabricating a new type of bioactive and mechanically reliable porous metal scaffolds-densified porous Ti scaffolds. The method consists of two fabrication processes, 1) the fabrication of porous Ti scaffolds by dynamic freeze casting, and 2) coating and densification of the porous scaffolds. The dynamic freeze casting method to fabricate porous Ti scaffolds allowed the densification of porous scaffolds by minimizing the chemical contamination and structural defects. The densification process is distinctive for three reasons. First, the densification process is simple, because it requires a control of only one parameter (degree of densification). Second, it is effective, as it achieves mechanical enhancement and sustainable release of biomolecules from porous scaffolds. Third, it has broad applications, as it is also applicable to the fabrication of functionally graded porous scaffolds by spatially varied strain during densification.

Bioactive and mechanically reliable metal scaffolds have been fabricated through a method which consists of two processes, dynamic freeze casting for the fabrication of porous Ti, and coating and densification of the Ti scaffolds. The densification process is simple, effective and applicable to the fabrication of functionally graded scaffolds.

Biometal systems have been widely used for biomedical applications, in particular, as load-bearing materials. However, major challenges are high stiffness ...

Synthesis of Keratin-based Nanofiber for Biomedical Engineering

Electrospinning, due to its versatility and potential for applications in various fields, is being frequently used to fabricate nanofibers. Production of these porous nanofibers is of great interest due to their unique physiochemical properties. Here we elaborate on the fabrication of keratin containing poly (&#949;-caprolactone) (PCL) nanofibers (i.e., PCL/keratin composite fiber). Water soluble keratin was first extracted from human hair and mixed with PCL in different ratios. The blended solution of PCL/keratin was transformed into nanofibrous membranes using a laboratory designed electrospinning set up. Fiber morphology and mechanical properties of the obtained nanofiber were observed and measured using scanning electron microscopy and tensile tester. Furthermore, degradability and chemical properties of the nanofiber were studied by FTIR. SEM images showed uniform surface morphology for PCL/keratin fibers of different compositions. These PCL/keratin fibers also showed excellent mechanical properties such as Young&#39;s modulus and failure point. Fibroblast cells were able to attach and proliferate thus proving good cell viability. Based on the characteristics discussed above, we can strongly argue that the blended nanofibers of natural and synthetic polymers can represent an excellent development of composite materials that can be used for different biomedical applications.

Electrospun nanofibers have a high surface area to weight ratio, excellent mechanical integrity, and support cell growth and proliferation. These nanofibers have a wide range of biomedical applications. Here we fabricate keratin/ PCL nanofibers, using the electrospinning technique, and characterize the fibers for possible applications in tissue engineering.

Electrospinning, due to its versatility and potential for applications in various fields, is being frequently used to fabricate nanofibers. Production of ...

Rapid Mix Preparation of Bioinspired Nanoscale Hydroxyapatite for Biomedical Applications

Hydroxyapatite (HA) has been widely used as a medical ceramic due to its good biocompatibility and osteoconductivity. Recently there has been interest regarding the use of bioinspired nanoscale hydroxyapatite (nHA). However, biological apatite is known to be calcium-deficient and carbonate-substituted with a nanoscale platelet-like morphology. Bioinspired nHA has the potential to stimulate optimal bone tissue regeneration due to its similarity to bone and tooth enamel mineral. Many of the methods currently used to fabricate nHA both in the laboratory and commercially, involve lengthy processes and complex equipment. Therefore, the aim of this study was to develop a rapid and reliable method to prepare high quality bioinspired nHA. The rapid mixing method developed was based upon an acid-base reaction involving calcium hydroxide and phosphoric acid. Briefly, a phosphoric acid solution was poured into a calcium hydroxide solution followed by stirring, washing and drying stages. Part of the batch was sintered at 1,000 &#176;C for 2 h in order to investigate the products&#39; high temperature stability. X-ray diffraction analysis showed the successful formation of HA, which showed thermal decomposition to &#946;-tricalcium phosphate after high temperature processing, which is typical for calcium-deficient HA. Fourier transform infrared spectroscopy showed the presence of carbonate groups in the precipitated product. The nHA particles had a low aspect ratio with approximate dimensions of 50 x 30 nm, close to the dimensions of biological apatite. The material was also calcium deficient with a Ca:P molar ratio of 1.63, which like biological apatite is lower than the stoichiometric HA ratio of 1.67. This new method is therefore a reliable and far more convenient process for the manufacture of bioinspired nHA, overcoming the need for lengthy titrations and complex equipment. The resulting bioinspired HA product is suitable for use in a wide variety of medical and consumer health applications.

This paper describes a novel method for the rapid manufacture of high quality bioinspired nanoscale hydroxyapatite. This biomaterial is of great significance in the manufacture of a wide range of innovative medical devices for clinical applications in orthopedics, craniofacial surgery and dentistry.

Hydroxyapatite (HA) has been widely used as a medical ceramic due to its good biocompatibility and osteoconductivity. Recently there has been interest ...

An Ultrasonic Tool for Nerve Conduction Block in Diabetic Rat Models

Nerve conduction block with a high intensity-focused ultrasound (HIFU) transducer has been performed in normal and diabetic animal models recently. HIFU can reversibly block the conduction of peripheral nerves without damaging the nerves while using an appropriate ultrasonic parameter. Temporary and partial block of the action potentials of nerves shows that HIFU has the potential to be a useful clinical treatment for pain relief. This work demonstrates the procedures for suppressing the action potentials of neuropathic nerves in diabetic rats in vivo using an HIFU transducer. The first step is to generate adult male diabetic neuropathic rats by streptozotocin (STZ) injection. The second step is to evaluate the peripheral diabetic neuropathy in STZ-induced diabetic rats by an electronic von Frey probe and a hot plate. The final step is to record in vivo extracellular action potentials of the nerve exposed to HIFU sonication. The method showed here may benefit the study of ultrasound analgesic applications.

This work presents the methodology of applying high intensity-focused ultrasound to block the action potentials of diabetic neuropathic nerves.

Nerve conduction block with a high intensity-focused ultrasound (HIFU) transducer has been performed in normal and diabetic animal models recently. HIFU ...

An Additive Manufacturing Technique for the Facile and Rapid Fabrication of Hydrogel-based Micromachines with Magnetically Responsive Components

Polyethylene glycol (PEG)-based hydrogels are biocompatible hydrogels that have been approved for use in humans by the FDA. Typical PEG-based hydrogels have simple monolithic architectures and often function as scaffolding materials for tissue engineering applications. More sophisticated structures typically take a long time to fabricate and do not contain moving components. This protocol describes a photolithography method that allows for facile and rapid microfabrication of PEG structures and devices. This strategy involves an in-house developed fabrication stage that allows for the rapid fabrication of 3D structures by building upwards in a layer-by-layer fashion. Independent moving components can also be aligned and assembled onto support structures to form integrated devices. These independent components are doped with superparamagnetic iron oxide nanoparticles that are sensitive to magnetic actuation. In this manner, the fabricated devices can be actuated using external magnets to yield movement of the components within. Hence, this technique allows for the fabrication of sophisticated MEMS-like devices (micromachines) that are composed entirely out of a biocompatible hydrogel, able to function without an onboard power source, and respond to a contact-less method of actuation. This manuscript describes the fabrication of both the fabrication set-up as well as the step-by-step method for the microfabrication of these hydrogels-based MEMS-like devices.

An additive manufacturing strategy for processing UV-crosslinkable hydrogels has been developed. This strategy allows for the layer-by-layer assembly of microfabricated hydrogel structures as well as the assembly of independent components, yielding integrated devices containing moving components that are responsive to magnetic actuation.

Polyethylene glycol (PEG)-based hydrogels are biocompatible hydrogels that have been approved for use in humans by the FDA. Typical PEG-based hydrogels ...

A Novel Surgical Technique As a Foundation for In Vivo Partial Liver Engineering in Rat

Organ engineering is a novel strategy to generate liver organ substitutes that can potentially be used in transplantation. Recently, in vivo liver engineering, including in vivo organ decellularization followed by repopulation, has emerged as a promising approach over ex vivo liver engineering. However, postoperative survival was not achieved. The aim of this study is to develop a novel surgical technique of in vivo selective liver lobe perfusion in rats as a prerequisite for in vivo liver engineering. We generate a circuit bypass only through the left lateral lobe. Then, the left lateral lobe is perfused with heparinized saline. The experiment is performed with 4 groups (n = 3 rats per group) based on different perfusion times of 20 min, 2 h, 3 h, and 4 h. Survival, as well as the macroscopically visible change of color and the histologically determined absence of blood cells in the portal triad and the sinusoids, is taken as an indicator for a successful model establishment. After selective perfusion of the left lateral lobe, we observe that the left lateral lobe, indeed, turned from red to faint yellow. In a histological assessment, no blood cells are visible in the branch of the portal vein, the central vein, and the sinusoids. The left lateral lobe turns red after reopening the blocked vessels. 12/12 rats survived the procedure for more than one week. We are the first to report a surgical model for in vivo single liver lobe perfusion with a long survival period of more than one week. In contrast to the previously published report, the most important advantage of the technique presented here is that perfusion of 70% of the liver is maintained throughout the whole procedure. The establishment of this technique provides a foundation for in vivo partial liver engineering in rats, including decellularization and recellularization.

A Novel Surgical Technique As a Foundation for In Vivo Partial Liver Engineering in Rat

We establish a novel surgical technique for an in vivo single liver lobe perfusion model in rat as a prerequisite for further studying in vivo partial liver engineering in the future.