Fig. 3: Terminalia sensory bristles regulate sequence progression.

a, Top: example video snapshot of abdominal bending. Scale bar, 1 mm. Bottom: brightfield image of the female posterior abdomen, approximating terminalia bristle surface contact (white box at top). The dashed blue line indicates the approximate substrate surface. Scale bar, 50 μm (b–e). b, Diagram of the female posterior abdomen (lateral aspect); orange circles, bristles innervated by GFP-labeled neurons in the representative ATB-1>mCD8–GFP image in c (left); gray circles, non-innervated bristles; T6–T8, sixth–eighth tergite; S6 and S7, sixth and seventh sternite; A, analia; OV, ovipositor valve. c, Representative images of the posterior abdomen from two of nine ATB-1>mCD8–GFP females (lateral (left) and ventral (right) aspects); mCD8–GFP expression, membrane of ATB neurons (green); autofluorescence, abdominal cuticle (magenta); background, overlaid brightfield images revealing extended bristles; orange boxes, regions shown in d. d, Higher-resolution regions of the left image in c displaying GFP-labeled and brightfield images. Red asterisks indicate bristles innervated by single GFP-labeled neurons. e, Representative image of the ventral nerve cord (left) and abdominal neuromere (right) from three ATB-1>mCD8–GFP females stained with anti-GFP (ATB neurons, green) and anti-bruchpilot (nc82; synaptic neuropil, magenta). Black bars flanking the left image indicate the region shown at higher resolution on the right. f, Average probability (P) of progression from bending to burrowing. Only flies that exhibited two or more bend bouts were considered (n = 17, 22, 15, 27, 17, 23, 25, 24 and 36 flies per group). Here and in g and i–k, box bounds indicate the 25th and 75th percentiles, the red line indicates the medians, and the whiskers indicate the 5th and 95th percentiles; o, data from individual flies; +, outliers. Here and in g and i, *P < 0.05, **P < 0.01 and ***P < 0.001; data were analyzed by two-sided Wilcoxon rank-sum test followed by a Bonferroni correction (Supplementary Table 7). g, Average probability of progression from burrowing to egg out. Only flies that exhibited two or more burrowing episodes were considered (n = 17, 22, 15, 27, 17, 17, 25, 24 and 41 flies per group). h, Representative ethograms depicting bending and burrowing behavior on 1.0% agarose. Each ethogram depicts data from five flies (n = 3 events per fly); <, eggs deposited on the wall; t = 0, egg out. i, Fraction of eggs deposited on walls of chambers containing 1% agarose substrate (n = 19, 33 and 32 flies per group). Only flies that released three or more eggs were considered. j, Average normalized depth of penetration of eggs released on substrates of varying firmness (GAL4-only, n = 10, 27, 21, 25, 29 and 19 flies per group; UAS-only, 29, 13, 34, 18, 24 and 12 flies per group; ATB-1>Kir2.1, 19, 10, 22, 24, 25 and 4 flies per group). Here and in k, *P < 0.05, **P < 0.01 and ***P< 0.001; data were analyzed by Kruskal–Wallis test with a post hoc Tukey’s HSD test (Supplementary Table 7). k, Number of eggs released in 4 h on substrates of varying firmness (GAL4-only, n = 13, 29, 28, 30, 42 and 28 flies per group; UAS-only, 31, 13, 35, 19, 33 and 27 flies per group; ATB-1>Kir2.1, 20, 10, 26, 27, 27 and 10 flies per group).