Female-released chemical signals are crucial clues for mate-searching males to locate and gain sexual receptivity of conspecific females. Abundant behavioral evidence indicates that female spiders release sex pheromones to guide mate-searching behavior of conspecific mature males. However, the chemical nature of spider pheromones is poorly understood. Females of the funnel-web spider, Allagelena difficilis, employ sit-and-wait tactics for mating. Field observations indicate that males leave their retreats to search for potential mates during the breeding season. Therefore, we investigated whether virgin females release a sex attractant to conspecific males and then explored the chemical nature of the female pheromone. Four fatty acids extracted from the female bodies (palmitic acid, linoleic acid, cis-vaccenic acid and stearic acid) constitute a multiple-component sex attractant to conspecific males in A. difficilis. Unexpectedly, mated females also produce the same fatty acids, but at trace levels. Two-choice experiments showed that males were significantly attracted by the blend of the four fatty acids in appropriate concentrations while avoiding the blend consisting of the same acids at very low concentrations, suggesting that mate-searching males are able to discriminate virgin females from mated females by the quantities of female-specific fatty acids in the funnel-web spider A. difficilis.
