Beyond sodefrin: evidence for a multi-component pheromone system in the model newt Cynops pyrrhogaster (Salamandridae)

Sodefrin, a decapeptide isolated from the male dorsal gland of the Japanese fire belly newt Cynops pyrrhogaster, was the first peptide pheromone identified from a vertebrate. The fire belly salamander and sodefrin have become a model for sex pheromone investigation in aquatically courting salamanders ever since. Subsequent studies in other salamanders identified SPF protein courtship pheromones of around 20 kDa belonging to the same gene-family. Although transcripts of these proteins could be PCR-amplified in Cynops, it is currently unknown whether they effectively use full-length SPF pheromones next to sodefrin. Here we combined transcriptomics, proteomics and phylogenetics to investigate SPF pheromone use in Cynops pyrrhogaster. Our data show that not sodefrin transcripts, but multiple SPF transcripts make up the majority of the expression profile in the dorsal gland of this newt. Proteome analyses of water in which a male has been courting confirm that this protein blend is effectively secreted and tail-fanned to the female. By combining phylogenetics and expression data, we show that independent evolutionary lineages of these SPF's were already expressed in ancestral Cynops species before the origin of sodefrin. Extant Cynops species continue to use this multi-component pheromone system, consisting of various proteins in addition to a lineage-specific peptide.