The Age of Female <i>Pardosa pseudoannulata</i> Affects the Attractiveness of Their Dragline Silk Sex Pheromones to Males

This study aimed to investigate whether the attractiveness of female dragline silk sex pheromones in the wolf spider Pardosa pseudoannulata varies with female age, and to identify the key chemical compounds responsible for male attraction. We conducted integrated behavioral, chemical, and electrophysiological analyses. Dragline silk was collected from females of three age groups (young, middle-aged, and old). Male courtship behaviors were assessed in response to silk exposure. Silk extracts were analyzed using gas chromatography coupled with electroantennographic detection and mass spectrometry. Candidate pheromone compounds were further evaluated via electroantennography and Y-tube olfactometer assays to confirm their behavioral relevance. Male spiders exhibited significantly stronger courtship responses, shorter latency, higher palpal drumming, and increased leg vibration, toward silk from young and middle-aged females compared to older females. Three compounds-oleic acid, n-heptacosane, and squalene-were consistently identified as key pheromone components across age groups. Their electrophysiological activity was concentration and solvent dependent, while behavioral attraction was confirmed under tested conditions. In behavioral assays, synthetic versions of these compounds at 0.01 µg/µL in mineral oil significantly attracted males, confirming their role as sex pheromone components. The findings demonstrate that female age significantly influences the chemical composition and attractiveness of dragline silk pheromones in P. pseudoannulata. Younger females produce more attractive pheromone blends, guiding male mate preference. The identification of oleic acid, n-heptacosane, and squalene as bioactive compounds provides new insights into spider chemical ecology and supports potential applications in eco-friendly pest management strategies.