为生物医学和生物工程研究建立章鱼生态系统
1Department of Biomedical Engineering,Michigan State University, 2Neuroengineering division, The Institute for Quantitative Health Science and Engineering,Michigan State University, 3Biomedical Imaging division, The Institute for Quantitative Health Science and Engineering,Michigan State University, 4Department of Radiology,Michigan State University
Summary
了解章鱼独特的生理和解剖结构可以极大地影响生物医学研究。本指南展示了如何建立和维护海洋环境以适应该物种，并包括最先进的成像和分析方法，以可视化章鱼的神经系统解剖结构和功能。
Abstract
生物医学研究的许多发展都受到发现支持不同物种特定功能的解剖学和细胞机制的启发。章鱼是这些特殊的动物之一，为科学家提供了对神经科学，机器人技术，再生医学和假肢领域的新见解。对这种头足类动物的研究需要建立复杂的设施，并为章鱼及其生态系统提供重症监护，这对项目的成功至关重要。该系统需要多个机械和生物过滤系统，为动物提供安全清洁的环境。除控制系统外，还需要专门的例行维护和清洁，以有效保持设施长期运行。建议通过改变水族箱的景观，加入各种猎物，并引入具有挑战性的任务，为这些聪明的动物提供丰富的环境。我们的结果包括MRI和全身自发荧光成像以及行为研究，以更好地了解他们的神经系统。章鱼具有独特的生理学，可以影响生物医学研究的许多领域。为他们提供可持续的生态系统是发现其独特能力的第一个关键步骤。
Introduction
生物医学研究和生物医学工程中的新概念通常受到确定生物物种所拥有的特定策略以应对环境和生理条件和挑战的启发。例如，了解萤火虫的荧光特性导致了新的荧光传感器的开发，可以报告其他模式生物的细胞活性1;识别藻类中由光激活的离子通道已导致细胞和时间特异性基于光的神经调节的发展2，3，4，5;在玻璃鲶鱼中发现根据地球磁场导航的蛋白质导致了基于磁性的神经调节的发展6，7，8，9，10，11;了解Aplysia中的虹吸反射有助于理解行为的细胞基础12，13，14。
研究人员继续通过利用非常规实验室物种所具有的独特优势和对生理功能的新观点来扩展当前的生物工程和系统发育工具箱。联邦机构正开始通过资助对不同物种进行的新工作来支持这些研究。
章鱼属动物具有独特的解剖学和再生能力，以及对其每个手臂的适应性控制，迷人的生物学家和工程师，以及来自社会各个角落的迷人观众是章鱼17。事实上，在过去的几十年里，章鱼的生理和行为的许多方面都得到了研究15，16，17，18，19，20，21，22，23，24，25，26.然而，分子和进化生物学，机器人技术，运动记录，成像，机器学习和电生理学的最新发展加速了与章鱼生理学和行为学相关的发现，并将其转化为创新的生物工程策略27，28，29，30，31，32，33，34，35，36，37，38，39.
在这里，我们描述了如何建立和维护章鱼饲养，这对来自不同背景，科学兴趣和目标的科学家和工程师来说既有趣又相关。尽管如此，我们的结果集中在章鱼在神经科学和神经工程研究中的应用。章鱼具有高度发达的神经系统，中枢脑有4500万个神经元，视叶有1.8亿个神经元，八个轴索和外周神经节有3.5亿个神经元;相比之下，狗的神经元数量相似，而猫只有其中的一半40。与脊椎动物神经系统不同，只有32K传出和140K传入纤维将章鱼大脑中的数百万个神经元连接到其手臂每条轴向索中的数百万个神经元40，41，42。这些相对较少的互连纤维表明，执行运动程序的大多数细节都是在轴索本身中执行的，强调了章鱼具有的独特分布的神经元控制。章鱼的手臂具有非凡的精细运动控制能力，使它们能够掌握诸如打开罐盖之类的操作技能，即使它们在容器内也是如此。这种高度发达的预制运动能力是头足类动物（章鱼、墨鱼和鱿鱼）所独有的43。
事实上，通过数亿年的进化，章鱼已经发展出一个非凡而复杂的基因组和生理系统43，44 ，激发了科学和工程领域的新发展和进步。例如，基于章鱼吸盘解剖结构的防水粘合剂贴片可以粘附在潮湿和干燥的表面上45;受章鱼迷彩皮肤启发的合成迷彩材料可以将平坦的2D表面转换为带有凹凸和凹坑的三维表面46。微型软体和自主机器人（即Octobots），将来可以作为体内的手术工具47;并且还开发了连接到类似坦克的机器人48 的手臂（即OctoArm）。许多种类的章鱼用于生物医学研究，例如， 寻常章鱼， 中华章鱼， 变种章鱼和 双章鱼 （O. bimaculoides）;O. vulgaris 和O. bimaculoides 是最常见的34，49，50。最近对不同章鱼基因组的测序使该属特别感兴趣，并为章鱼研究开辟了新的领域34，43，51，52。
在我们的设置中使用的O. bimaculoides是一种中型章鱼物种，于1949年首次发现，可以在从加利福尼亚中部到下加利福尼亚半岛南部的东北太平洋海岸的浅水区找到17。它可以通过眼睛下方地幔上的假眼点来识别。与巨型太平洋章鱼（Enteroctopus dofleini）和普通章鱼（O. vulgaris）相比，加州双斑章鱼（O. bimaculoides）的尺寸相对较小，开始时小于几厘米，作为幼年生长迅速。当在实验室内饲养时，成年地幔大小可以长到100厘米的平均大小，重达800 g53，54。章鱼在最初的200天内有一个快速的生长期;到那时，他们被认为是成年人，并在余生中继续成长55，56，57。章鱼可能是同类相食的，特别是当两性一起被安置在水箱内时;因此，它们需要单独存放在单独的储罐中58。
Protocol
所有动物研究均由密歇根州立大学机构动物护理和使用委员会（IACUC）批准。 1. 八达通罐设备设置 首先，获取将纳入海洋环境系统的水族馆的所有非生物材料，如 材料表所示。尺寸以英寸为单位提供。 在安装之前，用70%乙醇和去离子（DI）水清洗所有管道，管道和过滤系统部件。清洁时请勿使用肥皂或任何其他化学品。 放置一张13英寸x 49?…
Representative Results
我们研究中的所有动物都是从野外获得的，因此它们的确切年龄无法确定，它们在实验室中的停留时间是可变的。每天观察章鱼的状况。我们没有看到寄生虫，细菌，皮肤损伤或异常行为。动物的平均体重为170.38 +/- 77.25克。每只动物都栖息在自己的40加仑水箱中。一周内记录的罐的参数的平均±标准偏差为：pH 8.4 ±0.0，盐度34.06±0.61 ppt，温度18.7±0.75°C，氨0.11±0.14 ppm，亚硝酸盐0.25±0.14 ppm，硝酸盐…
Discussion
系统设置：
水族馆生态系统的发展方式是采用过滤和氧化水的机械和生物方法。该系统的过滤元件利用袜子过滤器，蛋白质分离器和定期清洁来保持氮气和氧气水平。更重要的是，我们还依靠海洋微生物来消耗危险的含氮化合物和其他生物废物，并通过光合作用过程给水充气。除了使用藻类之外，向水中添加氧气的其他方法是通过带有附加空气石的外部曝气器。在添加任何细菌之?…
Declarações
The authors have nothing to disclose.
Acknowledgements
这项工作得到了NIH UF1NS115817（G.P.）的支持。GP部分由NIH拨款R01NS072171和R01NS098231支持。我们要感谢Emit Imaging的Patrick Zakrzewki和Mohammed Farhoud在Xerra Imaging Platform上收集和可视化数据方面的帮助和支持。MSU 与布鲁克 Biospin 签订了研究协议。
Materials
| 1-3/4 in. Drill Bit | Home Depot | 204074205 | Glass cutting tool Part number:1 |
| 1" flow sensors | Neptune Systems | Local Dealer | Pipe with sensor to measure water flow Part number:2 |
| 1" Slip Bulkhead Strainer | Bulk Reef Supply | 207113 | Strainer for water leaving tank Part number:3 |
| 10 gallon tank | Pruss Pets | Local Dealer | Fiddler crab holding tank Part number:4 |
| 4 inch X 12 inch 200 Micron Nylon Monofiliment Mesh Filter Sock w/ Plastic Ring | AQUAMAXX | UJ41171 | Filter for large organic matter in sump Part number:5 |
| 40 gallon aquarium | Pruss Pets | Local Dealer | 4 Food aquarium tanks Part number:6 |
| 60g poly tanks – rectangle | Pruss Pets | Local Dealer | 2 Water Storage (salt and freshwater) Part number:7 |
| Active Aqua 1/10th HP Hydroponic or Aquarium Chiller 2018 Model | WayWe | 719574198463 | For cooling water continuously Part number:8 |
| ALAZCO 2 Soft-Grip Handle Heavy-Duty Tile Grout Brush | ALAZCO | B06W2FT5V5 | Tank Cleaning Part number:9 |
| Ammonia Testing Kit | Aquarium Pharmaceuticals | 33D | For water testing Part number:10 |
| Apex system WiFi | Neptune Systems | Local Dealer | System connection for off site monitoring Part number:11 |
| API Aquarium Test Kit | Amazon | B001EUE808 | For water testing Part number:12 |
| API Copper Test Kit | Amazon | B0006JDWH8 | For water testing Part number:13 |
| Aqua Ultraviolet Classic UV 25 Watt Series Units | Aqua Ultraviolet | A00028 | For removing bacteria leaving sump system Part number:14 |
| AquaClear 50 Foam Filter Inserts, 3 pack | Aquaclear | A1394 | Food Tank Carbon Filter Inserts Part number:15 |
| Aqueon QuietFlow LED PRO Aquarium Power Filter 30 | Aqueon | 100106082 | Food tank filtering units Part number:16 |
| Auto Top Off Kit (ATK) (Each includes 1 FMM module, 2 optical sensors and 1 float) | Neptune Systems | Local Dealer | For freshwater tank Part number:17 |
| Automatic top off from RODI (LLC) | Neptune Systems | Local Dealer | From water storage to octopus tanks Part number:18 |
| Banded Trochus Snail | LiveAquaria | CN-112080 | For algae bin Part number:19 |
| Chaetomorpha Algae, Aquacultured | LiveAquaria | BVJ-76354 | For algae bin Part number:20 |
| Clams – Live, Hard Shell, Cherrystone, Wild, USA Dozen | Fulton Fish Market | N/A | Live food Part number:21 |
| Classic Sea Salt Mix – Tropic Marin | Bulk Reef Supply | 211813 | Salt for tank water Part number:22 |
| Clear Masterkleer Soft PVC Plastic Tubing, for Air and Water, 3/4" ID, 1" OD | McMaster | 5233K71 | Cleaning tool Part number:23 |
| Continuum Aquablade-P Acrylic Safe Algae Scraper W/ Plastic Blade – 15 Inch | Marine Depot | 4C31001 | Cleaning tool Part number:24 |
| Copper Testing Kit | Aquarium Pharmaceuticals | 65L | For water testing Part number:25 |
| Curve Refugium CREE LED Aquarium Light | Eshopps | 6500K | Algae bin light Part number:26 |
| Eheim 1262 return pumps | EHEIM | 1250219 | Pump for storage tanks Part number:27 |
| Eshopps R-100 Refugium Sump GEN 3 | Eshopps | 15000 | Sump system Part number:28 |
| Ethyl Alcohol, 200 Proof | Sigma-Aldrich | 64-17-5 | Anesthesia Part number:29 |
| Extech DO600 ExStik II Dissolved Oxygen Meter | Extech | DO600 | Oxygen measurment Part number:30 |
| Fiddler Crabs; live; dozen | NORTHEAST BRINE SHRIMP | N/A | Live food Part number:31 |
| Filter Cartrages | Aqueon | 100106087 | Food tank filters Part number:32 |
| Florida Crushed Coral Dry Sand – CaribSea | Bulk Reef Supply | 212959 | Sedimate for bottom of tank Part number:33 |
| FMM module | Neptune Systems | Local Dealer | Controller for apex system Part number:34 |
| Fritz-Zyme TurboStart 900 – Fritz | Bulk Reef Supply | 213036 | Bacteria start Part number:35 |
| Hand Operated Drum Pump, Siphon, Basic Pump with Spout, For Container Type Bucket, Pail | Granger | 38Y789 | Water Hand Pump Part number:36 |
| High pH Testing Kit | Aquarium Pharmaceuticals | 27 | For water testing Part number:37 |
| Imagitarium Fine Mesh Net for Shrimp | Petco | 2580993 | Shrimp and fish transfer net Part number:38 |
| Leak Detection Kit (LDK) – Includes FMM module plus 2 ALD sensors | Neptune Systems | Local Dealer | Placed on floor to detect water Part number:39 |
| Lee`S Algae Scrubber Pad Jumbo – Glass | Marine Depot | LE12007 | Cleaning tool Part number:40 |
| Live rocks | Pruss Pets | Local Dealer | Habitat for octopus Part number:41 |
| Long Bottle Cleaning Brush 17" Extra Long | Haomaomao | B07FS7J7PN | Tank Cleaning Part number:42 |
| Magnesium chloride | Sigma-Aldrich | M1028-100ML | Euthanasia Part number:43 |
| Magnetic Probe Rack | Neptune Systems | Local Dealer | For holding apex sensor probes Part number:44 |
| Marine Ghost Shrimp | NORTHEAST BRINE SHRIMP | N/A | Live food Part number:45 |
| Marineland C-Series Canister Carbon Bags Filter Media, 2 count | Chewy | 98331 | For elevated copper levels Part number:46 |
| Nitra-Zorb Bag | Aquarium Pharmaceuticals | AP2213 | Absorbes nirtogen compounds Part number:47 |
| Nitrate Testing Kit | Aquarium Pharmaceuticals | LR1800 | For water testing Part number:48 |
| Nitrite Testing Kit | Aquarium Pharmaceuticals | 26 | For water testing Part number:49 |
| Pawfly 2 Inch Air Stones Cylinder 6 PCS Bubble Diffuser Airstones for Aquarium Fish Tank Pump Blue | Amazon | B076S56XWX | Aierating water Part number:50 |
| Penn Plax Airline Tubing for Aquariums –Clear and Flexible Resists Kinking, 8 Feet Standard | Amazon | B0002563MM | Tubing for connecting air pump to air stone Part number:51 |
| Plumbing with unions/valves plus 3/4" flex hose | Pruss Pets | Local Dealer | Water transport Part number:52 |
| PM1 module | Neptune Systems | Local Dealer | Power control module for apex Part number:53 |
| Protein skimmer | Reef Octopus | AC20284 | Removes biowaste from system Part number:54 |
| PVC Apex Mounting board, grommets, wire mounts | Neptune Systems | Local Dealer | Helps ensure organization for wires and tubing within system Part number:55 |
| PVC Regular Cement and 4-Ounce NSF Purple Primer | Amazon | Oatey – 30246 | For connecting PVC pipes Part number:56 |
| RODI unit | Neptune Systems | Local Dealer | RO Water Part number:57 |
| Salinity Probes | HANNA probes | HI98319 | Measures salinity of water Part number:58 |
| Seachem Pristine Aquarium Treatment | Sea Chem | 1438 | Provides bacteria that break down excess food, waste and detritus Part number:59 |
| Seachem Stability Fish Tank Stabilizer | Sea Chem | 116012607 | Seachem Stability will rapidly and safely establish the aquarium biofilter in freshwater and marine systems Part number:60 |
| Set of lexan tops | Pruss Pets | Local Dealer | Aquarium tank lids Part number:61 |
| Set of Various extended length aquabus cables | Neptune Systems | Local Dealer | Cables for Apex system Part number:62 |
| SLSON Aquarium Algae Scraper Double Sided Sponge Brush Cleaner Long Handle Fish Tank Scrubber for Glass Aquariums | Amazon | B07DC2TZCJ | Cleaning tool Part number:63 |
| Standard-Wall PVC Pipe Fitting for Water, 45 Degree Elbow Adapter, 3/4 Socket Female x 3/4 Socket Male | McMaster | 4880K189 | PVC pipe Part number:64 |
| Standard-Wall PVC Pipe Fitting for Water, 90 Degree Elbow Adapter, 1 Socket Female x 1 Socket Male | McMaster | 4880K773 | PVC pipe Part number:65 |
| Standard-Wall PVC Pipe Fitting for Water, Adapter, 1 Socket-Connect Female x 1 Barbed Male | McMaster | 4880K415 | PVC pipe Part number:66 |
| Standard-Wall PVC Pipe Fitting for Water, Straight Reducer, 2 Socket Female x 3/4 Socket Female | McMaster | 4880K008 | PVC pipe Part number:67 |
| Standard-Wall PVC Pipe Fitting for Water, Tee Connector, White, 1 Size Socket-Connect Female | McMaster | 4880K43 | PVC pipe Part number:68 |
| Standard-Wall Unthreaded Rigid PVC Pipe for Water, 1 Pipe Size, 10 Feet Long | McMaster | 48925K13 | PVC pipe Part number:69 |
| Standard-Wall Unthreaded Rigid PVC Pipe for Water, 3/4 Pipe Size, 5 Feet Long | McMaster | 48925K92 | PVC pipe Part number:70 |
| Structural FRP Fiberglass Sheet, 48" Wide x 96" Long, 1/4" Thick | McMaster | 8537K15 | Table top material Part number:71 |
| Structural FRP Fiberglass Square Tube, 10 Feet Long, 2" Wide x 2" High Outside, 1/8" Wall Thickness | McMaster | 8548K33 | Structural table materal Part number:72 |
| Tank Sediment | TopDawg Pet Supply | 8479001207 | Sediment for bottom of fiddler crab tank Part number:73 |
| Temperature probe | Neptune Systems | Local Dealer | Temperature probe for tanks Part number:74 |
| Tetra TetraMarine Large Saltwater Flakes for all Marine Fish | Amazon | B00025K0US | Fish, shrimp, and crab food Part number:75 |
| Tetra Whisper Aquarium Air Pump for 10 gallon Aquariums | Petco | 2335234 | Air pump for smaller tanks Part number:76 |
| Thick-Wall Through-Wall Pipe Fitting, for Water, PVC Connector, 1 Socket-Connect Female | McMaster | 36895K843 | PVC pipe Part number:77 |
| Vectra s2 pump | Bulk Reef Supply | 212141 | Aquarium Pump Part number:78 |
| Water Pump | TACKLIFE | GHWP1A | Pump for cleaning tanks Part number:79 |
| Wyze Cam v2 1080p HD Indoor WiFi Smart Home Camera with Night Vision | Amazon | B076H3SRXG | DeepLabCut Recording Part number:80 |
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VanBuren, T., Cywiak, C., Telgkamp, P., Mallett, C. L., Pelled, G. Establishing an Octopus Ecosystem for Biomedical and Bioengineering Research. J. Vis. Exp. (175), e62705, doi:10.3791/62705 (2021).