By differentially sampling the pheromone gland of females of the moth Heliothis virescens, we explored differences in pheromone on the surface, or outer distal layer(s) of the gland, and that located more proximally. For this, we used two sampling approaches, (i) a solid phase microextraction fiber rub followed by solvent extraction of residual pheromone (SPME rub/extract), and (ii) rapid solvent rinsing followed by solvent extraction of residual pheromone (rinse/extract). The SPME rub showed differences in component ratio between the dorsal and ventral gland surfaces. The rinse sampled a greater amount of pheromone than the SPME rub, sampling the whole gland surface as well as likely deeper into the gland. Compared to the other samplings, pheromone in the rinse was depleted in the minor component; consequently, the corresponding residual extract was highly enriched in the minor component. Further rinses of the gland yielded only small amounts of pheromone, with a similar component ratio as the first rinse, suggesting that the residual pheromone was less accessible and required extraction in solvent to be liberated. Sampling over the photoperiod showed that the more volatile minor component was depleted (relative to the major component) on the surface/outer cuticle over the period when females called. Together, these data suggest that the pheromone is stored, at least in part, on and in the gland cuticle and that distinct pools may be transported to different topographic regions. Females fed with a stable isotope tracer, incorporated label into pheromone in the gland very rapidly, with the labeled pheromone appearing on the gland surface ca. 1 min later.
Lepidoptera Noctuidae Solid phase microextraction Mass isotopomer distribution analysis Pheromone storage
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This work was funded in part by a United States Department of Agriculture Hatch Project ND02388 (to SPF). We thank the United States Department of Agriculture–National Institute of Food and Agriculture for an Instrument Grant, 2015-07238 (to SPF), contributing to the purchase of the GC/MS system. We are grateful to Professor Jérôme Casas for comments on an earlier version of this manuscript.
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