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Cardoza, et al.
nozzle and higher resolution printing head (Figure 2 To this end, we ran the same experiment, using a
right panel A, B, E, F). The extrusion is unhindered and 5 × 5 mm 3D-printed NGM square instead of a 20 ×
continuous during a 90° angle turn (example: Figure 2 20 mm one. Indeed, when the 20 × 20 mm square was
right panel E, framed area) and NGM lines can be laid used (Figure 5), the nematodes that crossed the barrier
successfully in 90° angles or smaller (Figure 7B in stayed inside the framed area for the remainder of the
Supplementary File). assay. In case of the 5 × 5 mm square (Figure 5 vs.
Figure 3C), nematodes did not remain inside the square
3.3. C. elegans ability to cross physical barriers once they enter; instead, they might enter, roam, exit,
and the effect of aging and even reenter. Due to this dynamic situation, the
number of worms counted inside the square did not
To confirm that Parnon-printed structures can be used increase monotonically, as shown in Figure 5, instead,
to investigate C. elegans behavior, we ran a series of it fluctuated. For this reason, and to reflect this dynamic
experiments. In the first scheme, we assessed nematodes’ behavior, instead of a time course (Figure 5D), results
ability to cross physical barriers, to reach a food source are presented as scatter plots (Figure 3A and 4B),
(Figures 3 and 5). We also examined the role of aging showing the mean and standard deviation of the
(Figure 5), feeding history, and prior experience percentage of worms scored inside the framed area at
(Figure 3). any given time point.
To establish that the 3D-printed squares constitute In addition, the 20 × 20 mm square (Figure 5)
a physical barrier for C. elegans, we conducted a control frames ~15% of the 60 mm plate surface area, whereas the
experiment, in which day 1 adult nematodes are allowed 5 × 5 mm one (Figure 3) frames <1% of that area. This
to reach a food source not framed by a physical barrier is probably why a smaller percentage of worms is scored
(Figure 5). Animals reached the food-containing area inside the 5 × 5 mm square at any given moment, compared
in large numbers quickly, and almost all of them (96 – to what happens with the 20 × 20 mm one. Therefore, the
100%) remained there for most of the 120 min assay relative size of the square with respect to the culture plate
(Figure 5D, gray circles). affects the dynamics of the assay and should be taken into
Next, we challenged day 1 adult C. elegans with a consideration when data are interpreted.
food source, framed by a 20 × 20 mm 3D-printed NGM
square (Figure 5). In this case, nematodes entered the 3.5. The effect of feeding history
framed area gradually and in lower rates, and at the end of When two groups of day 1 adults were tested (Figure 3A),
the 120 min assay, 74% of animals had crossed the square a FF one (FF; purple inverted triangles) and one that was
barrier and had reached the food source (Figure 5D, starved for 24 h prior testing (S; blue diamonds), it was
black circles). Therefore, the NGM square frame presents found that starved animals enter the food-baited square in
a physical barrier for nematodes. higher numbers than FF ones. This suggests that starved
We hypothesized that decision-making related to animals might have a stronger motivation to explore
physical challenges differs in young and old animals, their surrounding area and possibly to overcome physical
as aging-driven behavioral changes have been broadly obstacles as well.
reported in C. elegans [18-20] . To test our hypothesis, we ran When the experiment was performed with no OP50
the square barrier experiment with day 7 adult nematodes. inside the square (Figure 3A, woF – without food, shaded
In contrast to young adults, only 41% of day 7 C. elegans area, vs. wF – with food, non-shaded area), C. elegans
have crossed the barrier after 120 min (Figure 5D, behaved similarly, and starved animals (S woF, blue
purple diamonds). When day 7 adult nematodes were circles) entered the food-baited square in higher numbers
tested without a physical barrier (control experiment), than FF ones (FF woF, purple triangles). Therefore, the
results were similar to the ones of day 1 adult nematodes nematodes’ feeding history has a strong influence on their
(Figure 5D, pink diamonds). Since day 7 adults are tendency to explore beyond a physical barrier even if
beyond middle age, the above findings suggest an aging- there is no food beyond it.
related change in C. elegans physical barrier crossing
behavior. 3.6. The effect of prior experience

3.4. The effect of confined area size Laboratory populations of C. elegans are commonly
grown either in liquid cultures or on flat NGM plates
Next, we asked whether the size of the confined area with practically two-dimensional surfaces. C. elegans
and its ratio over the total available plate area affects the used in the present work have been cultured for many
dynamics of the assay, that is, whether worms travel in generations on NGM plates. Hence, we asked whether a
and out of the target area or stay inside the target square group of nematodes that have dwelled on a NGM plate
once they cross it. featuring a 3D-printed square will have familiarized

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