Establishing an Octopus Ecosystem for Biomedical and Bioengineering Research

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.