Dynamic Changes Of Antimictobial Peptides Secreted By Skin Of Rana Dybowskii Under Bacterial Stresses

Amphibians are the transitional species from the aquatic to the terrestrial. Their skin has gradually established a defensive natural immune defense system in the long-term evolution, to remove pathogenic microorganisms in the environment. But it has been found that amphibian populations were drastically reduced or disappeared global trend in recent years, and one of the important reasons of this phenomenon is infectious disease of amphibians. However, the relationship between environmental microorganisms and amphibian skin antimicrobial peptides is not clear.To reveal the changes of amphibian skin secretions under microbial challenges, the brown frog (Rana dybowskii) was selected as the test object, and the test subjects were divided into 6～72h,1～23d two observation periods.6～72h period, with 4 groups:E.coli bath group, E.coli sac injection group, S.aureus bacteria bath group and S.aureus sac injection group.1～23d period, after using a mild electrical stimulation emptying the secretion of skin gland, divided into E.coli bath group and LB medium group. Results showed by RP-HPLC analysis, protein concentration and antibacterial activity comparison. Observe the features and antimicrobial activity of skin secretions to reveal dynamics of secretion of amphibian skin against environmental microorganisms.The RP-HPLC results showed that normal skin secretions of R. dybowskii (control group) has isolated 26 peaks, indicating a strong broad-spectrum antimicrobial activity, particularly against G+ bacteria, and had low hemolytic activity.After challenged 6～72h, RP-HPLC peptide profiles showed no significant changes in the number of elution peaks, but peak C-K showed a specific change, in addition to peaks C, D and E showed fluctuation, the peak area of F, G and H significantly increased, peak I and K were significantly decreased. The antibacterial activity and protein concentration of skin secretions of R. dybowskii showed a time-dependent increasing trend.After challenged 1～23d, RP-HPLC peptide profiles displayed that 1～18d skin secretions significantly reduced and the number of elution peaks down to 4-10, restored to the level before challenged to 23d. And peaks A, B, C and D were persistent and had strong inhibition of G- and G+ bacteria. Peptide concentrations decreased significantly on day 1, and 1～7d E.coli bath groups were significantly higher than the LB bath groups, but 13-18d were opposite. Restoring the secretions of R. dybowskii to the level of unchallenged needed at least 23 days, as secretion still had antibacterial activity.This study indicated that antimicrobial peptides of skin secretions of R. dybowskii as the major components of skin immune defense system can induce non-specific immunity when facing different environmental stress, especially more sensitive to S.aureus. The results provide important theoretical and experimental basis for mode of skin secretions of R. dybowskii changes in further.