Abstract
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.
Highlights
(Fig. 1, indicated in dark and light green) from which the decapeptide pheromone can be cleaved (Fig. 1, indicated in dark green)
Expression analyses resulted in mapping of 17,607,281 of these reads on the transcripts that were obtained by RACE PCR, indicating that at least 17.4% of the total RNA sequencing (RNA-Seq) reads of the dorsal gland belong to the SPF-family
Despite the fact that sodefrin in the Japanese fire belly newt has served as a model for newt pheromone research since its discovery, the alongside secretion of multiple uncleaved SPF proteins has remained undetected for a long time
Summary
(Fig. 1, indicated in dark and light green) from which the decapeptide pheromone can be cleaved (Fig. 1, indicated in dark green). SPF transcripts have been PCR-amplified in the genus Cynops[22,23], information on effective translation, precursor abundance, and release of these proteins into the water during courtship is inexistent As a consequence, it is currently unknown whether Cynops species actively use full length SPF pheromones next to sodefrin, or whether the decapeptide has replaced the function of SPF in this genus. We first performed RNA-Seq transcriptome analyses of the male pheromone-producing dorsal gland to study the significance of SPF with regard to sodefrin transcript expression in this species To validate these results, we performed proteome analyses of water in which a male Japanese fire belly newt had been tail fanning, and demonstrate that multiple SPF proteins are effectively secreted and tail-fanned to the female during courtship (see movie S1). We combined a phylogeny of SPF-family transcripts with our transcriptome and proteome data to show that both alpha and beta SPF ‘s were already effectively secreted in water before the origin of the peptide sodefrin
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