A new candidate courtship pheromone in aquatically courting newts.

Males of advanced salamanders (Salamandroidea) are unique among vertebrates in having internal fertilization without copulation. Instead, sperm is transferred via an external spermatophore, which the female takes up with her cloaca. Since a successful transfer of sperm heavily relies on a female following the male track at spermatophore deposition, males have evolved various courtship displays to persuade a female in picking up the sperm. In several aquatically reproducing newts, males do not longer display physical contact during courtship, but instead rely on emitting chemosignals to the female. Previously, we isolated and experimentally tested protein pheromones of the Sodefrin Precursor-like Factor (SPF) family and demonstrated that they can elicit female courtship responses in a European newt. Here we combined proteomic analyses with transcriptome analyses (RNASeq) of the pheromone-producing gland to show that several European newt species additionally secrete multiple 15 kDa proteins in the water during courtship. These cysteine-rich proteins do not show any apparent relationship to other known proteins in vertebrates and are, similar to SPF, among the most highly expressed proteins in the pheromone-producing glands. This high expression, together with abundant secretion during tail-fanning in water, suggests that the 15 kDa proteins serve a courtship pheromone function in newts.