A Study On The Diversity Of Antimicrobial Peptides In The Skin Of Black-spotted Frog (Rana Nigromaculata) In Summer In Harbin, China

Antimicrobial peptides (AMPs) are an important component of innate immunity of amphibians due to their functions of broad spectrum of bactericide, viral and tumor resistance. Amphibians use different AMP profile selectively to cope with different pathogenic community in different environment. This indicates an essential role of pathogenic community in the evolution of AMPs and raises a question how amphibians choose AMP profiles in coping with different pathogenic community. There has not been definite answer to this question up to date. We analyzed AMP profiles of black-spotted frog (Rana nigromaculata) in June 2009 in Harbin, China. Adult frogs were captured in the wild and divided into two groups randomly. One group was killed in situ immediately and skin samples were collected and preserved in liquid nitrogen. The other group was transferred into the lab and sampled skins after being raised in clear water for three weeks. Profiles, physical and chemical properties and mode of selection of AMPs were compared between the two groups. Seventy one cDNAs from 65 genes encoding 22 species of AMPs belonging to 10 families were cloned in total. Seven families containing 17 species of AMPs encoded by 62 cDNAs belonging to esculentin-2, nigrocin-2, odorranoin-S1, temporin-1, pelophylaxin-2, odorranain-P1 and odorranain-B1 were shared by the two groups. Two families of 4 species of AMPs encoded by 8 cDNAs belonging to brevinin-1 and pelphylaxin-3 were unique to the wild group. One species of AMP encoded by 3 cDNAs that was identified in ranids for the first time and named nigromaculatin was only seen in the lab group. All shared AMPs were alkaline and cationic peptides with typical a-helix structure except for family nigrocin-2 and odorranoin-S 1 with random coil structure. Family nigromaculatin was acidic and randomly coiled AMP. Family temporin-1 contained 22 cDNAs evolving under negative selection and revealed the highest expression, a little higher in the wild group relative to the lab group. Family esculentin-2 contained 16 cDNAs evolving under negative selection as well or near neutral selection. But their expression was moderate. Family pelphylaxin-3 containing 6 cDNAs under positive selection was the second abundant AMPs in the wild group relative to family temporin-1. Family odorranain-B1, brevinin-1 and nigromaculatin were all under negative selection but with low expression. Analysis also indicated that the selection of AMPs was in a trend of changing from negative selection to positive selection with increment of genetic distance relative to their ancestral genes. These results suggested that (1) the black-spotted frog has at least 65 gene loci encoding AMPs; (2) there are stable components and environmental specific components in AMP profiles when the black-spotted frog is exposed to different environment. The genes of stable AMP components are primordial and conservative. Whereas, the genes of environmental specific AMP components are usually recent and evolving in positive selection; (3) In case of simplification and reduction of pathogenic community such as lab group was, AMP expression may reduce in both AMP species and their abundance, indicating the expression of AMPs follow economic principle.