After level three of the behaviotype tree, subjective visual inspection of the videos failed to
detect differences between related behaviotypes.
Nonetheless, they do have distinct properties for
some of the features for some of the time (Fig.
2D). Behaviotypes 17 and 18 represent animals
with tracking errors in which the MWT stream-
ing and sketching software inverted the front
and back of the animals (fig. S6 and movies S33
to S36). IDT assigned animals with this type of
tracking error to the two separate behavioral clus-
ters, conveniently isolating such errors.
Many of the optogenetically evoked, automatically detected behaviotype subfamilies are
similar to previously described larval responses
to various natural stimuli (10, 20, 21). IDT also
detected behavioral categories not previously
documented. This is unsurprising because only
Fig. 3. Learning neuron line–behaviotype maps via manifold testing.
(A) Behaviotype probability vectors for a number of lines. Each row shows the
percentage of animals performing a behaviotype for optogenetic activation of a
particular line: 30 randomly sampled test lines (top), negative controls (pBDPU-
ChR2) with no optogenetically activated neurons (middle), and positive controls—
nociceptive (ppk-ChR2, noci 1; R38A10-ChR2, noci 2); mechanosensory (iav-
ChR2); and proprioceptive (R11F05-ChR2) neuron lines with known effects on
behavior (bottom). (B) Line dissimilarity matrix showing pairwise P values for all
1054 lines computed via the manifold test. The first entry is the negative control.
Remaining entries are sorted according to P value for the comparison with the
negative control, from lowest to highest; 455 lines (4 positive controls + 451 test
lines) were significantly different from the negative control (hit lines), and 598
lines were not (nonhit lines). (C) The P values between all pairs of known
somatosensory neuron lines and negative controls are “correct”: Those that should
be significantly different according to previous studies are, and those that should
not be are not. This panel is a subset (top left 5 × 5 submatrix entries) of (B). (D)
Behaviotype probability distributions for all 451 significantly distinct test neuron
lines, sorted according to their maximum probability behaviotype. (E) The clusters
of lines that bias the probability toward the same behaviotypes are reproducible,
demonstrated by the fact that the mode of the empirical null distribution of
the adjusted Rand index (ARI) for reliable clustering (black curve) is lower in
all four repeated trials than the observed ARI (all P values < 0.01).