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

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.

Females of the four-eyed frog, Pleurodema thaul (Anura, Leptodactylidae), respond behaviourally to conspecific male scent

“…Kikuyama et al (1995), discovered that males of the fire belly salamander (C. phyrrogaster) release a female-attracting pheromone (denominated Sodefrina) and males of C. ensicauda release a peptide from their abdominal gland called Silefrin that attracted females (Yamamoto et al 2000). The fire belly salamander and their Sodefrin pheromone have become a model for the study of sex pheromones in aquatic salamanders, resulting in the discovery of a multicomponent pheromone system (Van Bocxlaer et al 2016).…”

Exaptation as a Mechanism for Functional Reinforcement of an Animal Pheromone System

“…The oldest known vertebrate sex pheromone system is the ''sodefrin precursor-like factor'' (SPF) pheromone system, a series of proteins involved in the courtship process of many salamander families (Plethodontidae, Ambystomatidae and Salamandridae) [9,[12][13][14][15][16][17]. In aquatically reproducing newts (Salamandridae), males display an underwater courtship dance in which they direct a stream of these water-soluble protein pheromones from their cloaca toward the female [18,19].…”

“…Total RNA (1 mg) was extracted from the pheromone-producing glands using the RNeasy Plus Universal Mini Kit (QIAGEN). Whole transcriptome shotgun sequencing (RNA-seq, paired-end) was performed on the pheromone-producing dorsal glands of one palmate newt male (Lissotriton helveticus, wild-caught; 52 Mio 50 bp read pairs at Baseclear, Leiden, the Netherlands; reads obtained earlier in [9]), two alpine newt males (Ichthyosaura alpestris, wild-caught; 48 Mio 50 bp read pairs at Baseclear and 57 Mio 100 bp read pairs at DNAVision, Gosselies, Belgium; reads obtained earlier in [17]), one southern crested newt male (Triturus karelinii, captive bred; 159 Mio 100 bp read pairs at DNAVision) and one Japanese fire belly newt male (Cynops pyrrhogaster, captive bred; 51 Mio 50 bp read pairs at Baseclear; reads obtained earlier in [13]). Dorsal glands form a dorsal part of the cloaca that secretes into the posterior end of the cloacal cavity.…”

Insights into the skin of caecilian amphibians from gene expression profiles

“…We annotated several candidate sequences expressed in the caecilian skin that could encode AMPs and SPF peptide pheromones. SPF proteins identified here in caecilians belong to the same gene family of sodefrin that is a courtship pheromone produced in the gonads of male salamanders [ 43 , 44 ]. Our results indicate the potential production of a multiple pheromone cocktail with high species-specificity in both male and female caecilians.…”