The Large-Scale Cultivation of Nematodes to Study Their Collective Behaviors

We are studying the mechanism underlying collective behavior from the perspective of active metaphysics and neuroscience. In the research field of collective behavior, experimental system has been established using non-living things and at the cellular level. However, it remains still challenging to design the animal labor experimental system to study the collective behavior in the laboratory.

We successfully reconstructed the collective behavior with traditionally used model animal, nematodes aeris. This system make it possible to control the parameter with mathematical model proposed to whole animal's collective behavior, and studies neural mechanism underlying collective behavior using molecular genetics. Begin by placing four adult worms on an nematode growth medium, or NGM, agar plate seeded with E.coli OP50.

Grow the first generation worms to starvation on the NGM plate at a temperature of 23 degrees Celsius for seven days. Next, transfer small amounts of dog food agar, or DFA medium, to the center of a new NGM plate seeded with E.coli OP50. For optogenetic experiments, pour 40 microliters of all-trans-retinal onto the DFA prior to worm inoculation.

Next, place 0.5 milligrams of dog food on the DFA medium, about two millimeters away from the plate lid. Avoid contamination by radiating the NGM plate with ultraviolet light for 15 minutes. Gather the starved worms from the NGM plates using sterilized water, then inoculate the gathered worms onto the DFA medium located on the NGM.

Propagate the worms at 23 degrees Celsius, enabling them to climb towards the plate lid over 10 to 14 days. Increased humidity exhibited larger compartment sizes of the worm network patterns before ultimately collapsing, leaving dormant worm clusters on the inner surface of the lid. Begin by placing a new NGM plate devoid of E.coli and DFA onto an aluminum plate on the stage of a macrozoom microscope.

Use a Pelletier temperature controller unit to maintain the plate bottom at 23 degrees Celsius temperature for at least five minutes. Next, replace the plate with the lid of a plate that the nematode worms have climbed up onto. Increase the temperature of the plate bottom to 26 degrees Celsius to change the interior humidity.

Now capture images of the inner surface of the plate lid at 20 frames per second using the microscope camera. Keep the ZX 899 worms on DFA, maintaining this condition for five minutes before illumination. Now illuminate the ZX 899 worms attached to a Petri plate lid maintained at 23 degrees Celsius.

Capture the images of the inner plate surface at 20 frames per second. Increased humidity exhibited larger compartment sizes of the worm network patterns before ultimately collapsing, leaving dormant worm clusters on the inner surface of the lid.