Love Hug-Functional Validation of Nuptial Pad-Secreted Pheromone in Anurans

Chemical communication is an important mode of communication in the courtship and breeding processes of amphibians. In caudates, multiple components of sexual pheromones have been identified and functionally verified. One of these pheromone systems is plethodontid modulating factor (PMF). In anurans, the pheromone called amplexin was found in nuptial pads of ranids and was considered a member of the PMF system, yet its bio-function has not been tested. In this study, we obtained 18 amplexin transcript sequences from nuptial pads of Nidirana pleuraden (Amphibia, Ranidae) by transcriptome sequencing and found that the proteins translated by these transcripts are diversified, hydrophilic, and relatively stable. We also acquired a N. pleuraden amplexin isoform with the highest expression level in the transcriptome analysis through the prokaryotic expression system. Using two different animal behavioral experimental settings, we have tested the bio-function of the recombinant PMF protein (rPMF) in N. pleuraden's reproduction and found that the rPMF does not attract females but shortens the duration of amplexus significantly. This is the first study to verify the function of the PMF pheromone in Anura, indicating the pervasiveness of chemical communication during breeding in amphibians.