Proteomic Analyses of Courtship Pheromones in the Redback Salamander, Plethodon cinereus

Abstract

The evolutionary success of plethodontid salamanders for ~100 MY is due partly to the use of courtship pheromones that regulate female receptivity. In ~90 % of plethodontid species, males deliver pheromones by "scratching" a female's dorsum, where pheromones diffuse transdermally into the bloodstream. However, in a single clade, representing ~10 % of Plethodon spp., males apply pheromones to the female's nares for olfactory delivery. Molecular studies have identified three major pheromone families: Plethodontid Receptivity Factor (PRF), Plethodontid Modulating Factor (PMF), and Sodefrin Precursor-like Factor (SPF). SPF and PMF genes are relatively ancient and found in all plethodontid species; however, PRF is found exclusively in the genus Plethodon - which includes species with transdermal, olfactory, and intermediate delivery behaviors. While previous proteomic analyses suggested PRF and PMF are dominant in slapping species and SPF is dominant in non-Plethodon scratching species, it was unclear how protein expression of different pheromone components may vary across delivery modes within Plethodon. Therefore, the aim of this study was to proteomically characterize the pheromones of a key scratching species in this evolutionary transition, Plethodon cinereus. Using mass spectrometry-based techniques, our data support the functional replacement of SPF by PRF in Plethodon spp. and an increase in PMF gene duplication events in both lineage-dependent and delivery-dependent manners. Novel glycosylation was observed on P. cinereus PRFs, which may modulate the metabolism and/or mechanism of action for PRF in scratching species. Cumulatively, these molecular data suggest that the replacement of pheromone components (e.g., SPF by PRF) preceded the evolutionary transition of the functional complex from transdermal to olfactory delivery.

Co-option and evolution of non-olfactory proteinaceous pheromones in a terrestrial lungless salamander

“…In all molecular and proteomic studies to date, we have demonstrated recruitment and co-option of multiple gene families followed by rapid evolution [23,25,26,28,44]. Specifically, our proteomic studies demonstrate large changes in pheromone composition between genera, even for those using the same delivery mode [28,45]. A key question remains is what factors allow a gene to be coopted.…”

“…Recent proteomic analyses in the redback salamander (Plethodon cinereus) revealed that, despite using the transdermal mode of delivery, its pheromone composition was principally composed of PRF and PMF, similar to that of P. shermani (which uses slapping delivery) [28].…”

From molecules to mating: Rapid evolution and biochemical studies of reproductive proteins

“…Initial pheromone characterization was performed for P. shermani using multiple types of high performance liquid chromatography (HPLC) to isolate individual proteins, which were then identified by MS analysis using a shotgun EST library as a reference [109,110]. Subsequent studies with D. ocoee and P. cinereus instead used de novo transcriptomes as reference databases, with amplification of identified candidates by RT-PCR and validation by Sanger sequencing [111,112]. Despite detection of SPF mRNA by RT-PCR in all three species, it was only observed proteomically in D. ocoee , where it comprised ∼30% of the total pheromone.…”

“…First, PRF was heavily glycosylated in P. cinereus and not P. shermani , with the loss of glycosylation resulting from mutation of two NXS/NXT N-glycosylation motifs and an alternative signal peptidase cleavage position which removes an O-glycan near the N-terminus. While glycosylation can affect various aspects of protein solubility, stability, and receptor specificity, its functional significance in this pheromone context remains to be determined [111]. Second, P. shermani expresses ∼5–10 times more PMF isoforms compared to P. cinereus (Fig.…”

Divergence of species‐specific protein sex pheromone blends in two related, nonhybridizing newts (Salamandridae)

“…Little is known on how protein pheromone blends evolve species‐specific effects and how sexual selection shapes protein pheromone evolution (Gomez‐Diaz & Benton, ; Peterson et al., ; Touhara, ; Wyatt, ,). Although recombination, alternative splicing and glycosylation can add a source of variation on top of the existing protein variation (Janssenswillen, Vandebergh, et al., ; Van Bocxlaer et al., ; Wilburn et al., ), there are essentially two ways to evolve species specificity for a given multiprotein pheromone system. On the one hand, each of the proteins themselves can evolve through the process of random mutation, selection or drift, leading to sequence divergence (Lemey, Salemi, & Vandamme, ), which can potentially change their shape and consequent interactions with receptors (Nakada et al., ; Wyatt, ; Yamamoto et al., ).…”