Courtship pheromones salamanders

The only other experimental attempt to evaluate the effects of male courtship pheromones in salamanders is work conducted with the terrestrial plethodontid, Desmognathus ochrophaeus (Houck and Arnold, in prep.). Courtship interactions for this species (described above) involve pheromone delivery from the male s mental gland, and the probable use of premaxillary teeth to introduce this pheromone into the female s circulatory system. In their experiment, Houck and Arnold had three experimental male groups (1) Each male had its mental gland surgically removed, (2) Each... 

Feldhoff R.C., Rollman S.M. and Houck L.D. (1999). Chemical analysis of courtship pheromones in a Plethodontid Salamander. In Advances in Chemical Signals in Vertebrates (Johnston R.E., Miiller-Schwarze D. and Sorenson P., eds.). Kluwer, New York, pp. 117-126. 

In the plethodontid salamander D. ocoee, courtship duration was reduced for male-female pairs in which the female received a protein signal from the 20-25 kDa fraction of the male courtship pheromone. We interpret this reduction in courtship duration as an increase in receptivity for females receiving the pheromone. 

The delivery of male courtship pheromones is widespread among plethodontid salamanders (Houck and Arnold 2003), and other courtship pheromones are being discovered for this group (Houck, Palmer, Watts, Arnold, Feldhoff and Feldhoff 2007). The mode by which these pheromones are transferred to the female apparently has been modified from delivery via diffusion into the circulatory system to delivery that directly stimulates vomeronasal receptors (Fig. 20.1 Houck and Sever 1994 Watts et al. 2004 Palmer et al. 2005 Palmer et al. 2007). The behavior patterns and morphologies associated with these two delivery modes often remain static for millions of years. In contrast, evolution at the level of pheromone signals is apparently an incessant process that continuously alters the protein sequence and composition of pheromones both within and among species (Watts et al. 2004 Palmer et al. 2005 Palmer et al. 2007). 

Houck, L.D. 1986. The evolution of salamander courtship pheromones. In D. Duvall, D. MiHler-Schwarze and R.M. Silverstein. (Eds.) Chemical Signals in Vertebrates, Vol. IV Ecology, Evolution, and Comparative Biology. Plenum Press, New York. Pp. 173-190. 

Houck, L.D., Bell, A.M., Reagan-Wallin, N.L. and Feldhoff, R.C. (1998) Effects of experimental delivery of male courtship pheromones on the timing of courtship in a terrestrial salamander, Plethodon jordani (Caudata Plethodontidae). Copeia 1998, 214-219. 

Houck, L.D. and Reagan, N.L. (1990) male courtship pheromones increase female receptivity in a plethodontid salamander. Anim. Behav. 39, 729-734. 

Palmer, C., Watts, R.A., Gregg, R., McCall, M., Houck, L.D., Highton, R. and Arnold, S J. (2005) Lineage-specific differences in evolutionary mode in a salamander courtship pheromone. Mol. Biol. Evol. 22, 2243-2256. 

Rollmann, S.M., Houck, L.D. and Feldhoff, R.C. (2000) Population variation in salamander courtship pheromones. J. Chem. Ecol. 26, 2713-2724. 

Courtship pheromones are not necessarily species specific. Pairs of the woodland salamander, Plethodon shermani, courted for an equally long time (about 35 to 50 minutes) whether male pheromone from the mental gland of conspecifics or the allopatric species P. montanus or P. yonahlosscc was present, even though the composition of the proteinacous pheromones (plethodontid receptivity factor of these three species differ considerably (Rollmann et al, 2003). 

Another salamander courtship pheromone, identified in 1999, is a protein. During courtship, males of the Appalachian woodland salamander, Plethodon jordani, (Plethodontidae), a terrestrial species, actively deliver a pheromone to the female. In the mating season, males develop a mental gland, located... 

Male courtship pheromones can increase the receptivity of the female. In the plethodontid salamander Desmognathus ochrophaeus the male courts the female hy scraping her dorsum with his specialized premaxillary teeth and swahs the same area with secretion from the mental gland on his chin, amounting to an injection. In an experiment, filter paper with an extract from the excised mental glands of males was placed on the dorsum of females. These treated females mated 28% (59 minutes) sooner than controls (Houck and Reagan, 1990). 

The terrestrial salamander Plethodonjordani applies courtship pheromone to the female hy ruhhing or slapping his mental gland directly on the female s nares. This way the pheromone can stimulate the vomeronasal organ and accessory olfactory system. Experimental application of mental gland extracts to the nares of females accelerated the time until active courtship started (Houck etal., 1998). 

Population variation in salamander courtship pheromones. Journal of Chemical Ecology 26 2713-2724. 

Amphibian pheromones have been previously isolated fh>m newt and salamander species, however, this peptide, which we have named splendipherin, is the first pheromone isolated fiom any anuran species. The delivery method of those previously isolated amphibian species are very clear. Sodefiin, the ten-residue peptide pheromone of Cynops pyrrhogaster and silefiin, the ten residue peptide pheromone of Cynops ensicauda are both sent through the water by the male newts by a vigorous shaking movement of the tail (Kikuyama et al., 1995 Yamamoto et al., 2000). The 20 kDa proteinaceous male courtship pheromone of Plethodon jordani is applied to the female s sldn by direct contact (Rollman et al., 1999). 

CHEMICAL ANALYSIS OF COURTSHIP PHEROMONES IN A PLETHODONTID SALAMANDER... 

The main effect of courtship pheromones is to alter female behavior in such a way that insemination is more likely to occur. In the giant danaine butterfly, for example, the male produces courtship pheromones from specialized structures termed hairpencils. The pheromone is released onto the female s antennae as the male hovers above her. Following reception of the pheromone, the female assumes an abdomen-curling posture that apparently facilitates copulation (Nishida, Schulz, Kim, Fukami, Kuwahara, Honda Hayashi, 1996). A variety of other arthropod species also have courtship pheromones (e.g., Birch, 1974 Boppre, 1984 Grant, O Connell Eisner, 1989). These specialized chemosignals are not known for any vertebrate group, however, with the exception of salamanders (Houck, 1986). 

Delivery styles vary greatly among species of salamanders, yet the only function demonstrated for salamander courtship pheromones has been to increase female receptivity. The change in receptivity is inferred on the basis of reduced courtship time as a result of pheromone delivery. Reduced courtship time has been demonstrated quantitatively for two species of terrestrial plethodontid salamanders. In D. ocoee (the species using an injection mode of delivery), courtship time was shorter when a female was treated with a pheromone extract versus an inactive (saline) control solution (Houck Reagan, 1990). In the plethodontid R jordani, pairs in which the female was pre-treated with courtship pheromones escalated to advanced stages of courtship more quickly on average than did pairs in which the... 

Chemical Analysis of Courtship Pheromones in a Plethodontid Salamander... 

Figure 1. Courtship pheromone delivery in the terrestrial salamander R jordani. Pheromone delivery occurs while the pair are in the tail-straddling walk (Arnold 1976). The male turns back towards the female and brings his submandibular courtship gland (arrow) in contact with the female s nares. The female typically lifts her head so that pheromone delivery is facilitated.

Figure 2. Analysis of courtship pheromones by SDS-PAGE gels. The courtship pheromone extracts from a terrestrial salamander R jordani) contain approximately 20 stained protein bands. Protein concentrations were standardized on gels, so band thickness and intensity reflect actual differences. Courtship gland extracts prepared in different years showed a high level of consistency among years, indicating their non-volatility and stability in response to temperature fluctuations (repetitive freezing and thawing). Top arrow indicates isoforms of the protein component Pj-22 bottom arrow indicates isoforms of protein component Pj-10. Lane 1 = protein standards, lane 2 = extract from 1994, lane 3 = extract from 1995, lane 4 = pooled extract from 1992—1995.

Salamander courtship pheromones produced by male P. jordani share the basic characteristics of substances known to affect female reproductive behavior. The R jordani extract was comprised primarily of glycoproteins, including highly stable and non-volatile... 

Table 1. Average amino acid (aa) composition for four of the major isoforms of Pj-22, a major protein component of the male courtship pheromone from the salamander, R jordanf...

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