Chemical Characterisation of the courtship water of Palmate newts (Lissotriton helveticus, Salamandridae)

After hibernation on land, European newts (Salamandridae, Amphibia) return to the water for courtship and breeding. Males develop several sexual characteristics such as a bright coloration and an extended crest on back and tail. The tail is used for fanning (underwater waving) and is predicted to direct pheromones from the male cloaca to the nose of the female in a complex courtship behaviour. Although this behaviour has been known for decades, the molecules expressed during waving and their exact function have never been demonstrated. We studied the courtship of palmate newts (Lissotriton helveticus) in an integrative approach, combining ethological tests, transcriptomics and proteomics. First, we tested the ability of complete courtship water (water in which males just fanned their tail) to attract females and obtained a positive behavioural assay. To further characterise the molecules responsible for this attraction, we then isolated peptides and proteins from the courtship water and used precursor cDNA information to fully characterise them. Our analyses identified multiple peptides and proteins up to 200 amino acids, showing that newt courtship water contains more than a single pheromone, and likely holds multiple kinds of chemical information.