(A) A method for extracting the attractive cue deposited on substrate. Larvae were allowed to crawl over the inner surface of a glass vial for 30 min. Larvae were removed and replaced by solvent. Treated solvent was deposited on one side of an agarose plate, and untreated solvent on the other. (B) The attraction of wild-type D. melanogaster larvae to extracts from larval-treated glass using hexane, acetone, or ethanol as solvent compared to control solvent. Data points are the preference index scores of individual wild-type larvae. Hexane and acetone extracts elicited similar levels of attraction as plates treated directly with larvae. Ethanol extracts did not. (C) Chromatograph from GC analysis of D. melanogaster larval hexane extracts. The major components are saturated fatty acids, fatty acid monoenes and dienes. Each peak is labeled as follows: (1) dodecanoic acid, (2) (Z)-5-tetradecenoic acid, (3) (Z)-7-tetradecenoic acid, (4) tetradecanoic acid, (5) (Z)-9-hexadecenoic acid, (6) hexadecanoic acid, (7) (Z,Z)-9,12-octadecadienoic acid, (8) (Z)-9-octadecenoic acid, and (9) octadecanoic acid, methyl ester. Small amounts of (Z)-9-tetradecenoic acid (*) were also detected between peaks (3) and (4). (D) Schematic of a multiple-larva assay to measure the attractive activity of individual compounds identified from larval residue. Test surfaces were coated in a checkerboard pattern with candidate molecules (green) or vehicle solvent (white). (E) A behavioral screen measuring the attraction of control (attP2;UAS-TNT, black), R58F10-GAL4;UAS-TNT (red), and R58F10-GAL4; UAS-IMPTV (blue) larvae to individual compounds found in the D. melanogaster larval extracts. Data points are the preference index scores of small groups (10-15 individuals) of larvae in response to 50 fmol/cm2 of candidate molecule. Compound treatments (ANOVA; F9,113 = 22.3, p < 0.0001) and genotype by compound interactions (2-way ANOVA; F1,232 = 17.7, p < 0.0001) displayed significant heterogeneity. Larvae were attracted to (Z)-5-tetradecenoic acid and (Z)-7-tetradecenoic acid. Silencing the synaptic activity of the sensory neurons expressing R58F10-GAL4 strongly inhibited the response to these molecules. Attraction was partially restored when driving the inactive TNT gene product, IMPTV. (F) The response of attP2;UAS-TNT (black), and R58F10-GAL4;UAS-TNT (red) larvae to 50 fmol/cm2 (Z)-5-tetradecenoic acid over time. (G) The timecourse of response to 50 fmol/cm2 (Z)-7-tetradecenoic acid. Solid lines represent the mean preference index score at each timepoint. Shaded areas represent ± one standard deviation. (H) Dose response curves of larval preferences to (Z)-5-tetradecenoic acid (orange), (Z)-7-tetradecenoic acid (green), and saturated tetradecanoic acid (grey). Shaded areas represent ± one standard deviation. (I, J) The preference index scores (I) and preference time course (J) of attP2;UAS-TNT (black), R58F10-GAL4;UAS-TNT (red), and R58F10-GAL4; UAS-IMPTV (blue) larvae in response to an equimolar mixture ((25 fmol:25 fmol)/cm2) of (Z)-5-tetradecenoic and (Z)-7-tetradecenoic acids.