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3D Arenas for C. elegans Behavior
Basic motor skills and chemotaxis remain (Figure 3B) suggest that C. elegans behavior with regard
functional through later stages of adulthood in C. elegans to physical challenges is affected by their prior experience
nematodes ; however, some decline in locomotion and familiarity with similar structures. We expect that this
[42]
speed and spontaneous locomotion has been effect will be stronger if the animals are grown in a 3D
[43]
reported [19,44] . The observed difference between day 1 and environment since hatching, and more 3D structures exist
day 7 adult nematodes (Figure 5) could be attributed to in their culture plate.
age-affected locomotion performance.
Results from dispersal assays have shown that 4.5. Spatial confinement of C. elegans behavior
young adult C. elegans move away from the original Given that in the above experiments (Figure 3),
position and spread into a wide area on the assay plate, C. elegans get in and out of the square frames without
but older adults are more likely to remain close to their much difficulty, and the localized egg laying behavior
original location . This agrees with our findings on day (Figure 4) can be attributed to the fact that the only food
[19]
7 adult nematodes, which do not spread over the square source available is located inside the 3D squares. This
barrier as their day conspecifics 1 (Figure 5). Dispersal makes them a preferable location for egg laying since this
over physical obstacles has not been previously assessed. way the newly hatched progeny has immediate access
Notably, a non-negligible part of the day 7 population to food. Indeed, it is known that egg laying is regulated
in study did cross the barrier, revealing a diversity in by a number of environmental conditions , leading to
[54]
individual C. elegans behavior and maybe a diverse spatial control of the egg laying behavior. Moreover,
[45]
impact of aging-related changes . the egg laying rate in the presence of abundant food is
[46]
It is reported that learning and memory decline earlier significantly higher than in the absence of food . The
[55]
in adulthood than other physiological operations [7,42,47] use of the 3D squares works well with localized food
and show signs of deterioration on day 7 or even availability, and it proves very effective when it comes to
[42]
day 5 of C. elegans adult life . It is possible that some restricting egg laying and potentially the hatching of L1
[7]
aspects of decision making, for example, aspects related larvae as well.
to overcoming physical challenges, are affected as well.
Moreover, a decrease in food-seeking exploration in older 5. Conclusion
nematodes is possible. This is the first reported attempt to explore C. elegans
Our findings on starved C. elegans (Figure 3A) are in ability to cross physical obstacles, made of the same
alignment with known starvation-induced modifications nematode-friendly material on which worms are cultured
of nematode behavior that improves their food location in the laboratory. A method that enables fabrication of such
likelihood. Indeed, after prolonged starvation, C. elegans structures, as is the prototype NGM extruding Parnon 3D
change their strategy to long range dispersal [48-51] . Even printer, was the necessary condition to achieve this. We
after 10 – 20 min away from food, neuronal plasticity can demonstrate that new tools and assays are not only the key
change, leading to global, instead of local, search behavior to answering biological questions but, most importantly,
in adult nematodes . Moreover, it has been shown that can often shape the very questions posed. We envision
[52]
food deprivation increases threat tolerance in C. elegans, the applications of 3D-printing NGM to enable artificial
which causes the worms to make “bolder” decisions . landscaping and development of diverse playgrounds for
[53]
Broadening the explored area, even if it includes some the exploration of C. elegans 3D behavior.
physical challenges, may be an additional manifestation
of behavioral changes triggered by food deprivation. Acknowledgments
Feeding history seems to impact obstacle crossing more
than the presence or absence of food inside the framed We thank Ao-Lin Hsu for the use of space and equipment,
area (Figure 3B). and Nikos Chronis for the use of space. We are grateful to
C. elegans nematodes used in the present work Zijun (Justin) Yuan for hydrogel testing, to Chris Pannier
have been cultured for many generations on NGM for technical advice, to Bill Kirkpatrick and Kent Pruss
plates, conforming with common practice in the field. for assistance with machining, to Alex Shorter for use of
Consequently, the 3D-printed squares are practically the Ultimaker3 3Dprinter, to Michael Solomon and Yufei Wei
first 3D structure they encounter, besides the unfriendly for rheometry advice, to Arthur Sinclair for rheometer
walls of the plastic Petri dish itself and the NGM chunk facilitation, and to Ruiming Lu for help with glass cutting.
often used in culture maintenance, when transferring Funding
nematodes into a fresh culture plate. In case of the latter,
C. elegans usually actively interact with the chunk, but This work was funded by the University of Michigan
they commonly just crawl away from it, since the chunk Office of Research (UMOR)-Faculty Grants and Awards
is flipped over the fresh NGM plate . Our findings Program (EG). EG is the recipient of a NIH-NIA K01
[6]
140 International Journal of Bioprinting (2022)–Volume 8, Issue 4