Figure 1: Bicyclus male wing pheromone: FA precursors and in vivo labelling.

(a) Wing FA composition in 4-day-old males B. anynana. Distribution of the FA-derived pheromone components (Z9-14:OH and 16:Ald) (n=10) and the potential immediate FA biosynthetic precursors (n=5) in forewings and hindwings. Bars represent s.e.m. Hexadecanal is almost exclusively present in hindwings, whereas the Z9-tetradecanol distributes equally across the two wings¹⁸. The identified FA precursors distribute across both wings. (b) Proportions of FA precursors in each wing relative to the total wing FA content (100%). (c–e) Analysis of pheromone production in male butterfly wings via biochemical in vivo labelling. Panels represent typical gas chromatograms from SIM, which records diagnostic ions according to the number of deuterium atoms in the precursor applied onto the wing androconia (FW: forewing, left columns; HW: hindwing, right columns) and shows incorporation of labelled FA precursors into pheromone components (16:Ald and Z9-14:OH). (c,d) The Z9-14:OH pheromone pathway. The native Z9-14:OH was monitored via its molecular ion at m/z 212 and the diagnostic ions at m/z 194 [M-18]⁺ or 31 [CH₂OH]⁺. The upper panels show deuterium incorporation in FW and HW androconia from D₃-16:acid (c) and D₉-Z11-16:acid (d) into Z9-14:OH, which was monitored by ions showing 3 or 9 a.m.u. more, respectively, than the native ion at m/z 194. Middle and lower panels display the corresponding chromatogram traces from negative labelling with D₃-14:acid and DMSO controls in each wing, respectively. (e) The 16:Ald pheromone pathway. The native 16:Ald was monitored by the diagnostic ions at m/z 222 [M-18]⁺ and 196 [M-44]⁺. The upper panels show incorporation in FW and HW androconia of the labelled D₃-16:acid into 16:Ald, monitored by a diagnostic ion showing 3 a.m.u. more than the native ion at m/z 196, whereas there is no deuterium incorporation when solutions containing D₃-14:acid or DMSO only are applied onto the wings.