Evaluating Methods of Obtaining Male Pheromone from Hymenochirus Sp. Using Analytical Chemistry

Male Hymenochirus sp. frogs are known to release pheromone that attracts females of the same species. Four methods for collecting secretions containing pheromone in Hymenochirus sp. were tested: norepinephrine injection, gonadotropin-releasing hormone injection, homogenization of gland tissue, and electrostimulation of the skin over the breeding gland area. The samples collected were analyzed using high-performance liquid chromatography (HPLC) and mass spectrometry. The HPLC chromatograph for the male norepinephrine sample contained a peak at 6.4 min that was not in the female norepinephrine sample HPLC chromatograph. The male norepinephrine sample mass spectrum had a peak of m/z 292.0 not in the female norepinephrine sample mass spectrum. Breeding gland homogenate mass spectra showed major peaks with m/z values of 203.9, 245.2, 484.8, and 558.9. The male norepinephrine sample mass spectrum contained peaks with the same m/z values, but the female norepinephrine sample did not. Serotonin and a novel truncated hymenochirin were found in male and female samples collected using norepinephrine or GnRH injection. HPLC results showed that electrostimulation of male frogs, as performed in this study, did not cause secretion of as many chemical compounds as the other methods. Electrical stimulation was done with a voltage of 0.75 V and stainless steel electrodes, instead of the 4.1 V and platinum electrodes used in other studies. The current produced was likely insufficient to stimulate the breeding gland. Behavioral testing was conducted testing female attraction to males injected with GnRH, and certain HPLC fractions of the male norepinephrine and breeding gland homogenate samples. No attraction was found to any of the tested stimuli. HPLC chromatograph and mass spectrum differences between the male and female norepinephrine-induced samples suggested a pheromone could be present. However, without a behavioral trial result showing female attraction to a collected sample, the presence of a pheromone was not definitively proven