Courtship pheromone-induced c-Fos-like immunolabeling in the female salamander brain.

Plethodontid salamanders display intricate courtship behaviors. Proteinaceous courtship pheromones were recently discovered in the submandibular (mental) gland of the male Plethodon shermani, the red-legged salamander. Behavioral studies showed that these male pheromones are delivered by direct contact to the female snout and modulate her receptivity during courtship. Previous reports demonstrated that experimental application of courtship pheromones activates vomeronasal sensory neurons in P. shermani. The present study investigated the CNS response to courtship pheromones in that species using immunocytochemical detection of the immediate-early gene product c-Fos. The results show that application of a male gland extract to females activated Fos-like immunolabeling in the extended vomeronasal amygdala of the accessory olfactory system, as well as in the preoptic area and ventromedial hypothalamus; regions of the brain known to mediate reproductive responses in vertebrates. The gland extract additionally activated Fos-like labeling in the raphe median, possibly indicating a serotonergic activation. Application of individual purified courtship pheromone proteins resulted in increases in Fos-like labeling in some of the regions activated by the complete submandibular gland extract, but the pattern of labeling was not as clear as that of the complete extract. Unlike other known vertebrate reproductive pheromones, courtship pheromones in P. shermani were effective only at a high concentration. This could result from the particular mode of pheromone transfer in that species, which involves sustained direct contact between male and female. It is concluded that salamander courtship pheromones exert their influence on behavior through the vomeronasal pathway and its direct projections to the preoptic and hypothalamic regions.

Proteinaceous pheromone affecting female receptivity in a terrestrial salamander.

A 22-kilodalton protein was isolated from the submandibular (mental) gland of the male terrestrial salamander, Plethodon jordani (family: Plethodontidae). This proteinaceous pheromone, termed plethodontid receptivity factor (PRF), was experimentally delivered to the female during courtship and shown to increase female receptivity. In most plethodontid salamanders, ovulation occurs weeks or months after insemination, so the pheromone-induced change in receptivity is the only known function of PRF. The messenger RNAs corresponding to isoforms of PRF were transcribed into complementary DNA, cloned, sequenced, and shown to have homology with cytokines of the interleukin-6 family. Pheromone activity would represent a previously unrecognized function for cytokines.

Courtship behavior and plasma levels of androgens and corticosterone in male marbled salamanders, Ambystoma opacum (ambystomatidae).

We measured plasma levels of testosterone, dihydrotestosterone (DHT), and corticosterone for male marbled salamanders (Ambystoma opacum) collected during the breeding season. Our goal was to ascertain whether steroid levels changed in response to particular reproductive behaviors or laboratory confinement. Six groups of salamanders were examined: (a) MIGRATING, males migrating toward the pond basin during the breeding season; (b) LABORATORY, males kept under confined conditions in the laboratory for 10 days; (c) LAB-FIELD, laboratory males that were later released into seminatural enclosures in the field; (d) COURTING, males from male-female pairs in which the male actively courted the female (and deposited at least one spermatophore); (e) SOLO, males that were individually isolated from conspecifics; and (f) MALE-MALE, males that were placed together in pairs, and in which one male actively courted the other male. In three groups (COURTING, SOLO, and MALE-MALE), salamanders were placed in containers for observation and each male was observed for at least 2 hr prior to a plasma sample being taken. Circulating levels of testosterone, DHT, and corticosterone did not differ significantly for males in these groups. The similarity of androgen levels among the three groups indicated a lack of behaviorally evoked change under experimental conditions designed to reveal a behavior-androgen response. Male A. opacum differ taxonomically from other amphibians showing a behavior-androgen response (three species of toads in the genus Bufo) and also lack amplexus and male-male combat during competition for mates. The effects of confinement were indicated by levels of testosterone and DHT in LABORATORY males that were significantly lower than average levels of males in the following groups: MIGRATING, LAB-FIELD, and MALE-MALE. We inferred that LAB-FIELD males, following their release to seminatural enclosures, were able to regain plasma androgen levels typical of migrating males. This increase is one of very few demonstrations for amphibians of an increase in androgen levels upon release from laboratory confinement. Levels of corticosterone did not differ significantly between males that were active in the field and males that were kept in the laboratory. The similarity of corticosterone levels among these groups differs from the typical pattern of elevated corticosterone and depressed androgen levels in captive amphibians. Maximal corticosterone levels in breeding male A. opacum may act differently than in other species in which chronic elevations inhibit the pituitary-gonadal axis.