Non-targeted Metabolomics Aids in Sex Pheromone Identification: a Proof-of-concept Study with the Triangulate Cobweb Spider, Steatoda Triangulosa.

Targeted metabolomics has been widely used in pheromone research but may miss pheromone components in study organisms that produce pheromones in trace amount and/or lack bio-detectors (e.g., antennae) to readily locate them in complex samples. Here, we used non -targeted metabolomics—together with high-performance liquid chromatography–mass spectrometry (HPLC–MS), gas chromatography-MS, and behavioral bioassays—to unravel the sex pheromone of the triangulate cobweb spider, Steatoda triangulosa . A ternary blend of three contact pheromone components [ N -4-methylvaleroyl- O -isobutyroyl- l -serine ( 5 ), N -3-methylbutyryl- O -isobutyroyl- l -serine ( 11 ), and N -3-methylbutyryl- O -butyroyl- l -serine ( 12 )] elicited courtship by S. triangulosa males as effectively as female web extract. Hydrolysis of 5 , 11 and 12 at the ester bond gave rise to two mate-attractant pheromone components [butyric acid ( 7 ) and isobutyric acid ( 8 )] which attracted S. triangulosa males as effectively as female webs. Pheromone components 11 and 12 are reported in spiders for the first time, and were discovered only through the use of non -targeted metabolomics and GC–MS. All compounds resemble pheromone components previously identified in widow spiders. Our study provides impetus to apply non -targeted metabolomics for pheromone research in a wide range of animal taxa.