Establishing an Octopus Ecosystem for Biomedical and Bioengineering Research

摘要

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.

摘要

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'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'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.

引言

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-neuromodulation^2,3,4,5; discovering proteins in glass catfish that navigate according to the Earth's magnetic field has led to the development of magnetic-based-neuromodulation^{6,7,8,9,10,11}; understanding the siphon reflex in Aplysia has been instrumental to understanding the cellular basis of behavior^12,13,14.

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' physiology and behavior have been studied over the past decades^{15,16,17,18,19,20,21,22,23,24,25,26}. 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 strategies^{27,28,29,30,31,32,33,34,35,36,37,38,39}.

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' brain to the millions of neurons in each of their arm's axial cords^40,41,42. 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'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 system^43,44 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' suckers can stick to wet and dry surfaces⁴⁵; a synthetic camouflaging material inspired by the octopus' camouflage 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 O. vulgaris and O. bimaculoides being the most common^34,49,50. The recent sequencing of different octopus genomes makes this genus of particular interest and opens new frontiers in octopus research^34,43,51,52.

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 Giant Pacific Octopus (Enteroctopus dofleini) and Common Octopus (O. vulgaris), the California Two-Spot Octopus (O. bimaculoides) 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 g^53,54. 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 life^55,56,57. Octopuses can be cannibalistic, especially when both sexes are housed together within a tank; therefore, they need to be housed individually in separate tanks⁵⁸.

方案

All animal studies were approved by the Institutional Animal Care and Use Committee (IACUC) of Michigan State University.

1. Octopus tank equipment set-up

1. 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.
2. Wash all tubing, piping, and filter system parts with 70% ethanol and deionized (DI) water prior to the installation. Do not use soap or any other chemicals when cleaning.
3. Place a fiberglass table 13 inch x 49 inch x ½ inch (Part #71) with four table legs made of carbon fiber and with the dimensions of 2 inch x 2 inch x 23 inch (Part #72). Attach the legs directly under the corners of the tabletop.
4. Below the top surface, between each of the table legs, place 2 inch x 2 inch long (Part #72) carbon fiber stabilization braces attached to the underside of the table and directly against the edge of the top shelf. Attach with screws another shelf with the same dimensions directly on the ground below the table. Let the pump (see Table of Materials) sit directly on the bottom shelf surface while the tank sits on the top surface. This system is shown in Figure 1.
   ​NOTE: Water output from the tank is gravity fed and all tubing, except the ones feeding in and out of the tank, need to be lower than the bottom of the tank to ensure maximum drainage head pressure.

Figure 1: Octopus tank setup. Water inlet and outlet (a). Three octopus tanks each with an area of 1.22 m x 0.3 m (b). Please click here to view a larger version of this figure.

5. Drill a single 1¾ inch hole, 2 inches from one of the sides of the tank, using glass cutting drill bits. The bottom of the water output suction screen will determine the elevation of the output hole as shown on the right side of Figure 2a. The water level will be determined by the suction screen and will need to be at least 6 inches from the top of the tank allowing for a water splash zone.
6. Use a PVC primer and cement to permanently connect the sections. To do so, first, slide the end of the intended male PVC pipe into the end of the female pipe. Place a piece of painters' tape on the outside of the male part that is still visible to prevent the primer and the cement from showing on the outside of the pipe. Separate the parts after taping and place a light coat of primer on the outside of the male pipe following the application of the cement in the same area.
7. Refit the male pipe into the female pipe, as soon as possible, after the application of cement and remove the tape. 24 h after the application of the primer and cement, wash out newly connected parts with DI water. For curing time look at the cement product for further directions.
   NOTE: Ensure the setup of all tubing and equipment is placed properly prior to using PVC primer and cement; pipe length requirements may vary.
8. Next, permanently connect the 1 inch outer diameter (OD) end of the suction screen to the 1 inch inner diameter (ID) end of the elbow joint. Connect the end of the elbow joint to straight PVC tubing (1 inch OD). Connect the other side of the straight tubing then to the 1 inch ID of the through-wall straight adapter female socket connect.
   NOTE: ID refers to the widest distance between the inside walls of the pipe. OD refers to the outside of the tubing width.
9. Permanently connect the through-wall straight adapter to a straight 4 inch long PVC pipe with a 1 inch OD (from step 1.8). This pipe will face out of the tank.
10. Permanently connect the straight pipe to the center of the PVC connector (1 inch ID Tee shaped; from step 1.9). Next, permanently connect two 6 inch long (Part #69) pipes (1 inch OD) to both the opposite ends of the tee connector-one facing directly up for the air release and the other directly down for water flow.
11. Permanently connect the downward extended straight pipe (from step 1.10) to a female socket barbed pipe (1-inch ID) straight adaptor. Attach a 36 inches long rubber tube (¾ inch ID) to the barbed pipe adaptor.
12. Place the cooling system between the water output tubing and the sump system.
13. Attach the ¾ inch barb fittings, that comes with the system, to the chiller unit's input and output ports. Put the rubber tubing (from step 1.11) on the inlet fitting of the chiller.
14. Connect a new piece of ¾ inch ID tubing (from step 1.13) from the chiller output (from step 1.12) to the inlet of the sump system as shown in Figure 2b.
15. Next, place the 4 inch x 12 inch sock filter, with pore size of 200 µm, into its designated area as shown in Figure 2. Also, as depicted in Figure 2, place the protein skimmer and the return pump into their appropriate areas. Along with the return pump, attach the automatic top off float valve to the inside wall of the pump area, 2 inches above the top of the pump's water inlet; do not block the pump from being removed from the tank, if needed.
16. Permanently connect a straight 12-inch-long tube (¾ inch OD) to the pump's outlet (from step 1.15). On the other end of the ¾ inch OD straight tube, permanently connect the tube's OD to a ¾ inch ID 45° elbow joint. To the other end of the joint, permanently connect a ¾ inch OD tubing.
17. Attach the other end of the straight tube (from step 1.16) to the 3/4 inch ID of a straight reducing adaptor. Permanently connect the larger adapter end (2-inch OD) to the input of the UV light.
    NOTE: Straight tubing lengths may vary.
18. Next, match the placement of UV light inlet with the pump's output pipe (from step 1.17) so that the pipe is not bending between light and pump (from step 1.15). Drill holes into the stabilization brace to match the UV light attachment holes. Match the size of screws with the drill bit and attach the UV light to the table using the screws given.
19. Permanently connect the 2-inch side of another reducing adaptor to the output of the UV light (from step 1.18). Attach a 1-inch OD of a 5-inch long straight tube to the adaptor's 1-inch ID. Next, connect a 90° corner piece with the 1-inch ID to the 1-inch OD tube; have the unattached end of the corner piece pointing toward the side of the tank where the water input is intended to go (same side as in step 1.5).
20. Permanently connect the other end of the corner (from step 1.19) to a 6 inches long tube (Part #69) having 1-inch OD with the input of the flow control unit (Part #2). Permanently connect another 1-inch OD tube (Part #69) to the output of the flow monitoring unit; the length must extend at least 3 inches beyond the side of the tank.
21. Using a 1¾ inch glass cutting drill bit (Part #1), cut a new hole 3 inches above the intended waterline and 2 inches away from the side of the tank (Figure 1a) on the side opposite to the one having water output hole. Attach another through-wall bulkhead fitting with a 1-inch slip (Part #77) facing out of the tank.
22. To the bulkhead slip connect a straight tube with the 1-inch OD and 4 inches length (Part #69) permanently. Cut down the tubing from the last part of step 1.21 to match the distance this tubing extends from the tank. Permanently connect a 90° tube (Part #65) to each of the open pipes and cut a final 1-inch OD straight tube (Part #69) that permanently connects both corner pieces.
    NOTE: Figure 3 shows a simple representation of the aquarium system.
23. Set up the rest of the control system (Part #34), first mounting the power strip (Part #53) to the table itself or to a nearby wall. Next to it mount the fluid monitoring module (Part #2).
24. Connect the flow sensor, power strip, and the leak detection sensors to the module. Set up the growth light (part #26) that is attached to the algae bin (Figure 2).
25. Plug in the flow sensor, UV light, growth light, pump, and protein skimmer to the energy bar. Set up the water control system programming according to the manufacturer's manual.
26. Prepare saltwater by mixing half a cup of commercially available salt mix with 1 gallon of reverse osmosis (RO) or deionized (DI) water. Make 45 gallons to fully fill one tank and sump system.
27. Turn on the pump within the sump system flow controller and keep adding saltwater until the automatic top off valve is in the off position so no additional freshwater is required.
28. Once the water is full, stop filling and turn on the water chilling unit to set the temperature between 18 °C to 22 °C as this is the preferable temperature range⁵³. Turn on the protein skimmer.
29. Add 30 kg of crushed coral to the bottom of the tank as well as a layer of crushed coral to the bottom of the algae bin. Add in multiple live rocks and any other additions to the octopus environment. Place a top to cover the opening of the tank.
    NOTE: Live rocks are dead coral that are inhabited by macroscopic marine life such as bacteria and algae.
30. Add nitrifying bacteria used in the saltwater aquariums as directed on the packaging. Keep adding this as directed, checking temperature, salinity, pH, ammonia, nitrite, and nitrate daily with water testing kits, pH sensor, and temperature sensor. Safe values for ammonia, nitrite, and nitrate levels are below 0.5 ppm, 0.25 ppm, and 10 ppm respectively⁵⁸.
31. Ensure UV light is turned off for the days nitrifying bacteria is being added to allow the saltwater microorganisms to grow. After parameters are within safe ranges, the UV light can be reactivated.
32. After the system is established, also check that the pH and oxygenation is at 8.0-8.4 and ⁵⁹, respectively. Prior to adding any animals to the aquarium, check for the presence of any copper and oxygen levels within the system using a copper water testing kit.
    ​NOTE: Copper causes damage to invertebrates and it interferes with osmoregulation in fish gills^60,61.
33. If copper is found in the water, test the DI/RO water source. After determining that the water source does not contain copper, perform a 30% water change and place the activated carbon block (Part #46) within water. If the problem persists, perform a full water change and clean all the parts.
34. After all the water parameters are determined to be within safe levels, add 10 ghost shrimp into the system at least a week prior to adding the octopuses. This will help introduce biomass for bacteria and indicate the overall water quality.
35. Add additional aquarium ecosystem inhabitants to the algae bin. This includes Chaetomorpha spp. (spaghetti algae), Trochus Sp. (banded trochus snail), and Mercenaria mercenaria (cherrystone clams).

Figure 2: Sump system. Side view of the sump system (a). Top view of the sump system (b). Please click here to view a larger version of this figure.

Figure 3: Aquarium with sump filtering system below the tank and environmental control units. Green arrows indicate direction of water flow through the system. Water flowing from section one to two for cooling and onto three to separate heavy biological matter from lighter matter. Heavy waste floats to the bottom and out to section five while the smaller biological matter flows into the sock filter within section four. Water flows from four underneath section five entering the protein skimmer in six to remove remaining waste within the water. The algae bin contains microorganisms to break down waste, ammonia, and nitrates as well as oxygenate the water. In the last part of the system, more water is added to account for evaporation prior to being pumped back into the tank. Please click here to view a larger version of this figure.

2. Storage tanks

1. Set up two tall 60-gallon water storage tanks, one for the saltwater and the other for RO water. Ensure that the freshwater tank's maximum fill line is taller than the table. Attach a ¼ inch tubing to the automatic top of the float valve in the sump system and attach the other end of tubing to the bottom of the freshwater tank.
   NOTE: This is to refill if water evaporates. Salt will stay in the water.
2. Fill the saltwater tank with water and add the proportional amount of salt to the tank. Continuously aerate the saltwater storage tank for mixing and proper oxygenation. Wait for an hour before use to ensure full mixing of the salt.
   ​NOTE: The saltwater tank is useful for refilling the tanks after cleaning.

3. Food tank setup

1. For keeping shrimp alive for longer than a week, store them in a separate tank from the octopus with the salinity below 30 ppt and the temperature close to 25 °C.
2. To do so, one week after the octopus tanks are matured, transfer 8 gallons of matured saltwater to the shrimp tank. Add 15 kg of crushed coral to the bottom of the tank. Add a few live rocks to the tank for hiding spots for molting (Figure 4).
   NOTE: Matured seawater refers to the process of allowing marine bacteria to grow within the saltwater as shown in step 1.30.
3. Attach a cannister filter to the edge of the tank. Set up the cannister filter as directed by the manufacturer. Add an air pump next to the tank connected to a tube with an attached air stone put into the tank.
4. Clean the filter and change the filter pads every week. Also, 25% of the water will need to be changed at the same time. Check nitrogen, pH, and temperature parameters daily in the food tanks with water testing kits as described in step 1.30. If water nitrogen parameters remain high, perform additional water changes and add a nitrogen absorbent bag to the water; or if problems persist longer than a month, the shrimp will need to be moved to a larger tank.
5. Add shrimp as soon as crushed coral sediment is dissipated. To add shrimp first, on arrival, move the shrimp without shipping water to the small saltwater tank for 5 min to remove biowaste. Then, the shrimp can be added directly to the tank. Mosquito fish, on arrival, can be added directly to the shrimp tank.
   NOTE: Shrimp and Mosquito fish can be purchased from any live animal commercial supplier listed on the material sheet or other food suppliers. It is also possible to offer octopuses defrosted shrimp.
6. Feed shrimp and fish with fish flakes, dead vegetation, or algae⁶², as directed on food instructions.
7. For the crab tank, add 1 gallon of saltwater and 10 kg of pebbles. Pile the pebbles on one side leaving dry land on one side and 2 cm of saltwater on the other side (as noted in Figure 4). The optimal environmental water parameters for these invertebrates should be 30-35 ppt and 22-25 °C for salinity and temperature^11,63, respectively.
8. Add fiddler crabs directly into the tank (Figure 4). Crabs will spend most of their lives on land but can be underwater for a few days at a time, making the tank that is partially underwater crucial for their long-term survival.
9. Feed fiddler crabs once a day by adding fish flakes into the dish on the dry area of the tank. Clean weekly by removing crabs and changing 100% of the saltwater. Clean the pebbles.
10. Store marine bivalve mollusks (clams and mussels) within the saltwater tanks for the octopuses to open themselves and provide another water filtering mechanism⁶⁴.
11. Place mussels inside a separate unoccupied tank for the first week to avoid placing an unnecessary waste load on the octopus tank's filtering system.
    ​NOTE: While the mussels have been the octopus' food of choice, they are more likely to die soon after arrival and will substantially increase the biological waste within the tank if they are present in large quantities.

4. Introduction of octopus to the tank

1. Ensure ammonia, nitrite, and nitrate levels are below 0.5 ppm, 0.25 ppm, and 10 ppm respectively. Have water hand pump available to remove octopus ink from the tank. It is also recommended to have two people for this procedure.
2. On arrival, place the bag on the scale and subtract the weight of the bag after the octopus is removed. Add an air stone to the bag to increase the water oxygenation while transferring the animal to their tank. Measure the shipping water's temperature and salinity. Record cases of prolonged illness after shipment.
   1. 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 down the sink. Add the same amount of water from the tank to the bag. Repeat every 10 min until the water temperature in the bag is no more than 1° different than the water temperature in the tank.
   2. Once the temperature difference of the bag and the tank are within 1°, ensure gloves are worn to move the octopuses to their individual tank. To move, 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.
   3. 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 approximate weight of the animal.
3. For the first 2 weeks after arrival, monitor the octopus' daily consumption that should be around 4% to 8% of their weight^58,65,66. The octopus should be checked on four times a day; this can be decreased to twice per day after 2 weeks. Weigh every two weeks to adjust their food consumption as needed.
   NOTE: Some species of octopus are known to escape from their tank, so it is advisable to place a 2.5 kg weight on the lid of their tank.

5. Daily care

1. Using a commercially available saltwater testing kit for pH, ammonia, nitrite, and nitrate, 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 colorimetric reactant to the corresponding tube.
2. If ammonia, nitrite, and nitrate levels are above 0.5 ppm, 0.25 ppm, and 10 ppm 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 fresh saltwater.
   NOTE: The above steps reduce nitrogen compounds within the ecosystem.
3. Remove all dead crab and shrimp carcasses from the tank as well as any octopus fecal matter using a hand pump. Remove all the remaining living crabs from the tank and move them back to the storage tank. Next, rearrange large objects within the tank.
4. Introduce half the number of the crabs that the octopus would eat daily to the tank weighing 1.25 +/- 0.25 g. 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.
   NOTE: The octopuses daily food consumption is 4%-8% of their weight⁶⁷. Frozen shrimp can also be provided as a food source based on the octopus' weight.
5. Offer five ghost shrimp daily. On an average, three were consumed in this experiment. 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.
   NOTE: Giving the animals a variety of food is not required and can prevent animals from being enticed by food during experiments. The feeding schedule used here to best monitor octopus feeding and behavior is to introduce half the number of crabs based on weight and increasing the number of shrimp to five in the morning. In the evening, introduce the second half of the crabs to the tank.

6. Weekly sanitation

1. Shut down the skimmer, pump, and algae bin lights prior to cleaning the sump system. Then, turn off the automatic valve of the system prior to removing water. Finally, remove the skimmer and all the water only from the sump system.
2. Lightly scrub algae bin to remove most of the biomass from its walls. Clean the rest of the sump area with a brush. Remove the sock filter, clean out with vinegar, and let it dry; rotate with another sock filter each week replacing with new ones every three months. Remove and clean out biomass from the top of the skimmer weekly.
   NOTE: Avoid using metal to clean the plastic as it will create scratches that could be prone to microbial growth.
3. Put the skimmer back into the system and begin refilling with saltwater. When the pump area is beginning to fill, all the systems can be turned back on. Stop adding water when the automatic top of the float valve is in the off position.

Figure 4: Tank for fiddler crabs (Minuca pugnax). The bottom of the tank is half designated for dry bed and the other half for 2 cm of shallow saltwater. Please click here to view a larger version of this figure.

Figure 5: Tank for ghost shrimp (Palaemonetes paludosus). Rocks in the shrimp tank provide places for the shrimp to hide and molt as well as for the growth of microorganisms. Please click here to view a larger version of this figure.

7. Care of unwell animals

1. Follow the guide reference⁶⁶ to assess octopus wellness.
   NOTE: For female octopuses, end of life cycle normally begins after laying eggs. The animal will begin to decrease food consumption and will stop eating altogether and will become more lethargic. Lifespan after the end-of-life process varies. No further action can be taken except feeding and monitoring the animal. Senescent males will decrease food consumption and become lethargic⁶⁸.

8. Octopus anesthesia

1. Perform octopus anesthesia as detailed in Butler-Struben et al.⁶⁹.
2. Obtain a 6 L container with lid that is at least 15 cm tall. Place 4 L of water directly from the octopus's tank into the container and provide aeration for 4 L of saltwater using a small air pump with air stone to disseminate oxygen to the water environment⁵⁸.
3. Prior to the octopus introduction, add 1% EtOH to the container. Before handling the octopus, record the number of breaths per minute by counting the exhalation of water from the siphon.
   NOTE: For octopuses within the researcher's laboratory, the baseline respirations is 16 - 24 breaths per minute.
4. Prior to moving the octopus, record the octopus' skin pigmentation and baseline breathing rate. Remove the octopus from the tank using a clean 4 L open mouth container by scooping it up with its surrounding water.
   NOTE: During anesthesia, breathing rates do not necessarily indicate complete anesthesia.
5. Weigh the octopus while in the container, and then move it by placing both hands around the octopus' body and lifting it up. A second person may be needed to remove the suctioned limbs from the container walls.
6. Quickly move the octopus into the prepared container with 1% EtOH. Close the lid to prevent a possible escape.
7. Record the respiration of the octopus per minute by counting the exhalation of water from siphon at the end of the first 5 min. If the respiration remains above baseline and the animal continues to respond to a light pinch, add an additional 0.25% EtOH to water. The addition of ethanol to water can continue to a maximum of 3% EtOH.
   NOTE: One indication that the octopus is unconscious is its loss of control of its chromatophores. In this case the skin appears paler than normal. A further indication is to lightly pinch the arms and test whether there is a motor response. If there is still no response at this point, the octopus is unconscious, and experiments can be performed.
8. While under anesthesia, monitor the octopus' breathing and color to ensure it remains unconscious for the duration of the procedure. If the octopus begins to awaken during the procedure, add an additional 0.25% EtOH.
9. For reversing the effects of ethanol anesthesia, transfer the octopus to a new 4 L or greater tank of oxygenated water from its permanent holding tank. Once the respirations return to normal, the octopus becomes active, and its skin returns to normal pigments; it can be moved back to its tank.

9. Octopus euthanasia

1. Follow the international standards for octopus euthanasia as detailed in Fiorito et al., Moltschaniwskyj et al., and Butler-Struben et al^57,58,69.
2. Prepare a new 6 L container with 4 L of water from the octopus' holding tank. Mix in MgCl₂ to a concentration of 4% to the euthanasia tank. Perform steps from 8.1 to 8.9 to anesthetize the octopus.
3. Move the octopus after step 8.8 to the euthanasia tank. After the breathing stops, wait for 5 min and perform a decerebration of the octopus or keep in the euthanasia tank for 5 additional minutes.

10. Behavior of O. bimaculoides

1. Do not feed the octopus on the mornings when they will be trained to use a screw cap container. Set up a camera recording device pointing at the area intended for feeding.
2. Obtain a 50 mL screw cap tube with 1 mm diameter holes throughout the surface and the cap for water flow throughout the container. Place a fiddler crab within the container. Place a weight within the container or attached to the outside for it to remain at the bottom of the tank.
3. Place the container at the bottom of the tank within the open area and in sight of the octopus and the camera. If the crab has not been eaten after 4 h, then remove it from the tube and resume the feeding schedule for the day. Keep performing this exercise daily.
   NOTE: This is shown in Figure 6 and discussed in the representative result section.

11. Octopus MRI

NOTE: Previously, evoked functional MRI responses in the octopus's retina were measured in anesthetized animals⁷⁰. Here, we obtained an ultra-high spatial resolution MRI of the octopus' nervous system that required hours of scanning. Thus, this was performed in a euthanized O. bimaculoides.

1. Obtain MRI images using a 7T system. Wrap the octopus in a kitchen-grade polyvinyl chloride plastic wrap to maintain the hydration of tissue. Place the octopus on the wrap, tuck in the ends, and then roll to seal.
2. Use a volume transmit/receive coil with a 4 cm diameter to acquire images of the brain and multiple arms. Use T1 weighted RARE sequence with the following parameters: Repetition time (TR) of 1500 ms, echo time (TE) of 20 ms, 117 x 117 x 500 µm resolution, 100 averages, RARE factor 8. These are typical MRI parameters for imaging rodent brains. Using a RARE factor makes the imaging faster, while 100 images are averaged together to increase the signal-to-noise ratio⁷¹.
3. Image the octopus arm using an 86 mm volume transmit coil and a 4 x 4 cm 4-channel array receive coil. Cut off an arm using surgical scissors and place it in a 15 mL conical tube filled with phosphate-buffered saline.
   NOTE: The sequence was a T1_weighted inversion recovery sequence (MP-RAGE) with parameters: TR/TE = 4000/2.17 ms, inversion delay 1050 ms, 100 x 100 x 500 µm resolution, 9 averages, scan time 1.5 h (Figure 7). An inversion-recovery sequence nulls the signal from water and increases contrast within the image; this sequence was chosen because it allows visualization of the internal anatomy of the arm⁷².

12. Cryo-fluorescence tomography (CFT) imaging

1. Flash-freeze the octopus: Work in a fume hood. Cover the bottom of a Dewar with dry ice, and then fill with hexanes. Slowly lower the octopus into the hexanes over about 10 min, adding fresh hexanes and dry ice as required to fully cover the octopus with cold hexanes. Keep the octopus frozen at -20 °C until it is embedded.
2. Embed and section the octopus: Create a rectangular mold of the appropriate size to hold the octopus using the tools provided by the CFT manufacturer. Cover the bottom of the mold with OCT (optimal cutting temperature) media (standard material used in histology laboratories) and let it freeze to a semi-solid gel.
3. Place the frozen octopus into the gel layer of the OCT, and then cover slowly with OCT in 2-3 layers. Between pouring steps, freeze the block steps until the OCT is at the gel stage. After the octopus is entirely covered, freeze the block for at least 12 h at -20 °C.
4. Load the sample into the cryo-fluorescence tomography system⁷³.
5. Section and image the entire euthanized O. bimaculoides at mesoscopic resolution using 3 emission/excitation filters thereby producing several 3D isotropic datasets.
6. When the sectioning reaches the arm and digestive system, transfer the sections to the slides for further histology.
7. Load the raw dataset into the reconstruction software from the CFT vendor specifically designed to enable fast processing.
8. Reconstruct a 3-dimensional stack using landmark alignment, histogram balancing, and fluorescence corrections and normalization, including the removal of subsurface fluorescence effects for each wavelength.
9. Once the final 3D stack is produced by the reconstruction tool, visualize the data with the imaging software tool and create fly-throughs with white light and fluorescence overlays along with 3D Maximum Intensity Projections (3D-MIPS), e.g., Figure 8⁷³.

结果

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 ± standard deviation for the parameters recorded for a tank over a week were: pH 8.4 ± 0.0, salinity 34.06 ± 0.61 ppt, temperature 18.7 ± 0.75 °C, ammonia 0....

讨论

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...

材料

本文使用的材料清单

姓名	公司	目录编号	评论
1-3/4 英寸	钻头 家得宝	204074205	玻璃切割工具 零件号：1
1" 流量传感器	Neptune Systems	本地经销商	带传感器测量水流的管道 零件号：2
1" 滑动隔板过滤器	散装礁石供应	207113	离开水箱的过滤器 零件号：3
10 加仑水箱	Preuss Pets	当地经销商	提琴蟹储水箱 零件号：4
4 英寸 X 12 英寸 200 微米尼龙单滤网过滤袜带塑料环	AQUAMAXX	UJ41171	过滤器，用于集水坑中的大量有机物 零件号：5
40 加仑水族箱	Preuss Pets	当地经销商	4 食品水族箱 零件号：6
60g 聚乙烯水箱 - 矩形	Preuss Pets	当地经销商	2 储水（盐水和淡水） 零件型号：7
Active Aqua 1/10th HP 水培或水族箱冷却器 2018 型号	我们	719574198463	用于连续冷却水 零件型号：8
ALAZCO 2 软握柄重型瓷砖灌浆刷	ALAZCO	B06W2FT5V5	水箱清洁 零件型号：9
氨测试套件	水族馆药品	33D	用于水质测试 零件型号：10
Apex 系统 WiFi	Neptune Systems	本地经销商	用于场外监测的系统连接 零件型号：11
API 水族馆测试套件	Amazon	B001EUE808	用于水质测试 零件号：12
API 铜缆测试套件	Amazon	B0006JDWH8	用于水质测试 零件型号：13
Aqua Ultraviolet Classic UV 25 瓦系列装置	Aqua Ultraviolet	A00028	用于去除离开集水坑系统的细菌 零件型号：14
AquaClear 50 泡沫过滤器插件，3 件装	Aquaclear	A1394	食品罐碳过滤器插件 零件型号：15
Aqueon QuietFlow LED PRO 水族馆电源过滤器 30	Aqueon	100106082	食品罐过滤装置 零件型号：16
自动加注套件 （ATK） （每个包括 1 个 FMM 模块、2 个光学传感器和 1 个浮子）	Neptune Systems	淡水	箱本地经销商 零件型号：17
从 RODI （LLC） 自动加注	Neptune Systems	当地经销商	从储水到章鱼缸 零件型号：18
带状 Trochus 蜗牛	LiveAquaria	CN-112080	用于藻类箱 零件型号：19
毛形藻类，水产养殖	活体水族馆	BVJ-76354	藻类箱 零件号：20
蛤蜊 - 活的，硬壳的，樱桃石的，野生的，美国 十几个	富尔顿鱼市场	不适用	活食 零件号：21
经典海盐混合物 - 热带马林	散装珊瑚礁供应	211813	水箱水盐 零件号：22
透明 Masterkleer 软 PVC 塑料管，用于空气和水，3/4" 内径， 1" OD	McMaster	5233K71	清洁工具 零件号：23
Continuum Aquablade-P 丙烯酸安全藻类刮刀带塑料刀片 - 15 英寸	船用仓库	4C31001	清洁工具 零件号：24
铜测试套件	水族馆药品	65L	用于水质测试 零件号：25
Curve Refugium CREE LED 水族馆灯	Eshopps	6500K	藻类仓灯 零件型号：26
Eheim 1262 回流泵	EHEIM	1250219	储罐泵 零件型号：27
Eshopps R-100 避难所集水槽 GEN 3	Eshopps	15000	集水槽系统 零件型号：28
乙醇，200 证明	Sigma-Aldrich	64-17-5	麻醉 零件型号：29
Extech DO600 ExStik II 溶解氧计	Extech	DO600	氧气测量 零件号：30
只招潮蟹;活体;十几	东北盐水虾	N/A	活食 零件号：31
个滤芯	Aqueon	100106087	食品罐过滤器 零件号：32
佛罗里达州碎珊瑚干沙 - 加勒比海	散装珊瑚礁供应	212959	水箱底部的沉积物 零件型号：33
FMM 模块	Neptune Systems	顶点系统本地经销商	控制器 零件型号：34
Fritz-Zyme TurboStart 900 - Fritz	散装珊瑚礁供应	213036	细菌 start 零件型号：35
手动鼓泵，虹吸管，带出水口的基本泵，用于容器类型桶， Pail	Grainger	38Y789	水手动泵 零件号：36
高pH值测试套件	水族馆制药	27	用于水质测试 零件号：37
Imagitarium 虾细网	Petco	2580993	虾和鱼转移网 零件号：38
泄漏检测套件 （LDK） - 包括 FMM 模块和 2 个 ALD 传感器	Neptune 系统	当地经销商	放置在地板上检测水 零件号：39
Lee'S 藻类洗涤器垫巨型 - 玻璃	船用仓库	LE12007	清洁工具 零件号：40
活石	Preuss 宠物	章	鱼 零件号：41
长瓶清洁刷 17" 超长	好毛毛	B07FS7J7PN	水箱清洁 零件编号：42
氯化镁	Sigma-Aldrich	M1028-100ML	安乐死 零件型号：43
磁探针架	Neptune Systems	本地经销商	用于固定顶级传感器探头 零件型号：44
海洋鬼虾	东北盐水虾	不适用	活食 零件型号：45
Marineland C 系列罐式碳袋过滤介质，2耐	嚼	98331	铜含量升高 零件号：46
Nitra-Zorb 袋	水族馆制药	AP2213	吸收氮化合物 零件号：47
硝酸盐测试套件	水族馆制药	LR1800	用于水质测试 零件号：48
亚硝酸盐测试套件	水族馆制药	26	用于水质测试 零件号：49
Pawfly 2 英寸气石圆筒 6 个气泡扩散器气石，用于水族馆鱼缸泵		B076S56XWX	充气水 零件号：50
Penn Plax 水族馆空气管 –清晰而灵活 抗扭结，8英尺标准	Amazon	B0002563MM	用于将气泵连接到气石的管道 零件型号：51
带接头/阀门的管道加上 3/4" 软管	Preuss Pets	当地经销商	水运 零件号：52
PM1 模块	Neptune Systems	当地经销商	apex 的电源控制模块 零件号：53
蛋白质撇渣器	Reef Octopus	AC20284	从系统中清除生物废物 零件型号：54
PVC Apex 安装板、索环、电线支架	Neptune Systems	当地经销商	帮助确保系统内电线和管道的组织 零件型号：55
PVC 普通水泥和 4 盎司 NSF 紫色底漆	Amazon	Oatey - 30246	用于连接 PVC 管道 零件型号：56
RODI 装置	Neptune Systems	本地经销商	RO Water 零件号：57
盐度探头	HANNA 探头	HI98319	测量水的盐度 零件号：58
Seachem 原始水族馆处理	Seachem	1438	提供分解多余食物、废物和碎屑的细菌 零件号：59
Seachem Stability 鱼缸稳定器	Seachem	116012607	Seachem Stability将在淡水和海洋系统中快速安全地建立水族箱生物过滤器 零件型号：60
lexan顶部	Preuss Pets	当地经销商	水族馆水箱盖 零件型号：61
各种延长长度的水上总线电缆	Neptune Systems	Apex系统本地经销商	电缆 零件型号：62
SLSON 水族箱藻类刮刀双面海绵刷清洁器玻璃水族箱长柄鱼缸洗涤器	亚马逊	B07DC2TZCJ	清洁工具 零件型号：63
标准壁 PVC 水管接头，45 度弯头适配器，3/4 插座母 x 3/4 插座公	McMaster	4880K189	PVC 管 零件型号：64
标准墙 PVC 水管接头，90 度 弯头 适配器， 1个插座女性x 1个插座男性	McMaster	4880K773	PVC管 零件号：65
标准-壁挂式 PVC 水管接头，适配器， 1 Socket-Connect 女性x 1 带刺男性	McMaster	4880K415	PVC 管 零件号：66
标准-壁厚 PVC 水管接头，直 减速机 2插座女性 x 3/4 插座女性	McMaster	4880K008	PVC 管 零件号：67
标准-壁厚 PVC 水管配件，三通接头， 白色， 1 尺寸 Socket-Connect 女性	McMaster	4880K43	PVC 管 零件号：68
标准-壁厚 无螺纹硬质PVC水管，1管尺寸，10英尺长	McMaster	48925K13	PVC管 零件编号：69
标准壁无螺纹硬质PVC水管，3/4管径，5英尺长	McMaster	48925K92	PVC管 零件编号：70
结构FRP玻璃纤维板，48"宽 x 96"长， 1/2" 厚	McMaster	8537K15	桌面材料 零件号：71
结构 FRP 玻璃纤维方管，10 英尺长，2" 宽 x 2" 高，1/8" 壁厚	McMaster	8548K33	结构台材料 零件号：72
水箱沉积物	顶部Dawg 宠物用品	8479001207	提琴蟹水箱底部的沉积物 零件号：73
温度探头	Neptune Systems	本地经销商	水箱温度探头 零件型号：74
Tetra TetraMarine 大型盐水片，适用于所有海洋鱼类	亚马逊	B00025K0US	鱼、虾和蟹食 零件型号：75
用于 10 加仑水族馆的 Tetra Whisper 水族箱气泵	Petco	2335234	用于较小水箱的气泵 零件型号：76
厚壁穿墙管件， 水用， PVC 接头， 1 个插座连接母	头 McMaster	36895K843	PVC 管 零件型号：77
Vectra s2 泵	散装礁石供应	212141	水族泵 零件型号：78
水泵	TACKLIFE	GHWP1A	清洗水箱泵 零件型号：79
Wyze Cam v2 1080p 高清室内 WiFi 智能家居摄像头夜视	Amazon	B076H3SRXG	DeepLabCut 录音 零件型号：80