Extended Data Fig. 7: Evidence against flies using spatial information in substrate search and against a feeding-on-higher-sucrose related explanation for substrate preferences in our free behavior chambers, alongside controls for the egg-laying rate function.

a, Schematic of a fly searching for an egg deposition site in a 0 vs. 500 mM chamber. ∆T_0mM and ∆T_500mM are all the intervals of time that a fly spent on 0 or 500 mM, respectively, during an egg-laying search period. ∆T_{last_500mM} is the last transit interval through 500 mM for eggs deposited on 0 mM. If a fly were positionally avoiding sucrose, ∆T_500mM would be less than ∆T_0mM. If a fly were to use spatial information during the search period—by taking a shortcut to get to the preferred 0 mM substrate at the end of a search—∆T_{last_500mM} would be less than ∆T_0mM and ∆T_500mM. If a fly were feeding on the higher sucrose substrate—and pausing as flies do when they feed⁸²—∆T_500mM would be larger than ∆T_0mM. b-d, ∆T_{lower_sucrose}, ∆T_{higher_sucrose}, and ∆T_{last_higher_sucrose} distributions for three different sucrose choice chambers. ∆T_{higher_sucrose} is not less than ∆T_{lower_sucrose} suggesting that flies are not positionally avoiding the higher sucrose option. ∆T_{last_higher_sucrose} is not detectably smaller than ∆T_0mM or ∆T_500mM suggesting that flies are not taking a shortcut—and thus not manifesting use of spatial information—at the end of the search. It is possible that flies use spatial information to guide the search in conditions with visible landmarks or where they perform less thigmotaxis (edge-hugging); our flies largely edge-hugged as they traversed the chamber. All experiments in this study were conducted in darkness. Note that our time-domain model for egg laying (Fig. 4a) could be readily augmented with spatial knowledge in that flies could putatively use their spatial sense to control which substrate they visit which would then impact their egg-laying drive. ∆T_{higher_sucrose} is not larger than ∆T_{lower_sucrose} indicating that flies are not pausing only on the higher sucrose substrate. We interpret this result to mean that flies are not suppressing egg deposition because of extensive feeding on the sucrose substrates. In addition, we did not notice additional proboscis extension on higher sucrose when we spent hours inspecting each video to annotate the egg deposition times. Note that our flies were very well fed before entering the chamber, which could have minimized this effect (Methods). 771 eggs from 17 flies (18 flies tested and 1 did not lay eggs), 1863 eggs from 42 flies (47 flies tested and 5 did not lay eggs), and 1345 eggs from 30 flies (30 flies tested), respectively. e, Mean egg-laying rates during the search period after a fly transitions across the plastic barrier in a single-option chamber, meaning that there is either 0 mM sucrose on both sides, 200 mM sucrose on both sides, or 500 mM sucrose on both sides. 90% confidence interval shaded. Egg-laying rates on the three different sucrose concentrations are similar in single-option chambers. The slightly higher egg-laying rates on lower sucrose is consistent with a possible, slight, innate preference for lower sucrose, which interacts with a much more prominent relative-value assessment of sucrose that governs egg laying rates (Fig. 3f–h). 895 eggs from 23 flies (24 flies tested and 1 laid no eggs), 1253 eggs from 27 flies (27 flies tested), and 528 eggs from 16 flies (17 flies tested and 1 laid no eggs) for 0 vs. 0, 200 vs. 200, and 500 vs. 500 mM chambers, respectively. f, Mean egg-laying rate during the search after a fly transitions across a mock vertical line. 90% confidence interval shaded. Same data as in panel e. The 5–10 s bin in this analysis has a higher egg laying rate than in the analysis from panel e, suggesting that part of the delay in egg laying after a transition is due to flies not laying eggs on the plastic barrier. g, Mean locomotor speed with ± s.e.m. shaded. A ~3 s delay exists between when a fly pauses and bends its abdomen to lay an egg till when an egg is deposited. This ~3 s latency is at least part of the reason why even the data in panel f do not show high egg laying rates in the 0–5 s bin. Analyzing the same data as in panels e-f.