Sex pheromones mediate mate location in many animal taxa. Widow spider males are attracted to sex pheromones disseminating from female webs. Upon arrival on a web, males court in response to web-borne contact pheromone components. N-3-Methylbutanoyl-O-methylpropanoyl-L-serine methyl ester (1) is the single known, weakly effective, contact pheromone component of female western black widows, Latrodectus hesperus. Moreover, the seasonal periodicity of pheromone signalling by female spiders is unknown. We tested the hypotheses that females of L. hesperus (1) deposit multiple contact pheromone components on their web that transition to mate-attractant pheromone components, and (2) increase pheromone signalling during the primary mating season. Analyses of web extract by gas and liquid chromatography-mass spectrometry (GC-MS; LC-MS) revealed N-3-methylbutanoyl-O-methylpropanoyl-L-serine (7), the corresponding acid of 1. Web extract of unmated female L. hesperus, and the synthetic blend of 1 and 7, were equally effective in eliciting courtship by males but web extract induced more sustained courtship. Tested singly, 7 prompted longer courtship behaviour by males than 1. Synthetic isobutyric acid (10), the hydrolysis product of 1 and 7, attracted male spiders in a field experiment. The abundance of 1 and 7 on female webs, with expected corresponding dissemination of 10 from webs, peaked during the summer when males are most abundant, indicating strategic sexual signalling by female spiders.

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