Transcriptomic insight into molecular processes of light-induced rapid darkening of non-obligative cave dweller Oreolalax rhodostigmatus (Megophryidae, Anura) and their genetic basis of pigmentation strategy

O. rhodostigmatus are non-obligative cave dwellers, whose tadpoles keep albinistic phenotype in caves but rapidly darken in light within 24 hours. Their coloration system is an excellent model for exploring the processes and mechanisms of light-induced pigmentation and revealing the genetic adaptation for non-obligative cave dwelling due to complicated life history. Using comparative transcriptomics, we found that melanocyte MCC (including melanogenesis and melanocytes proliferation) was responsible for the rapid skin darkening in was activated in O. rhodostigmatus. Light exposure induced robust activation of growth signals (including growth factor signals, MAPK signal pathways and PIK3-Akt signal pathways) at transcriptional levels, which were likely the upstream activation signals of melanocyte MCC in O. rhodostigmatus tadpoles. These results evidenced that amphibians and mammals likely share similar regulatory signals for light-induced melanocyte MCC. The conservation of pigmentation mechanisms across lower vertebrates and mammals imply that knowledge based on simpler models could also provide implications for causations and therapies of human pigmentation disorders and pigmented tumors. In aspect of genetic adaptation, an in-frame deletion of four amino acids in the membrane/extracellular junctions of the second and third transmembrane domains of O. rhodostigmatus MC1R, the receptor for melanogenesis signal, was identified. This mutation increases the negative charge of the ligand pocket of MC1R and results stereo-tandem of three aspartate residues aligning towards its ligand pocket. The ligand pocket of O. rhodostigmatus MC1R resembles a trap for positively charged ligands (a-MSH and ACTH) and likely increases the ligands-dependent activity of MC1R, providing an explanation for the rapid MCC of O. rhodostigmatus in light. Meanwhile, increased negative charge of ligand pocket likely decreased the constitutive activity of MC1R, in in supporting the albinistic phenotype of cave dwelling tadpoles. Therefore, genetic change of MC1R explains, at least to some extent, how the pigmentation system of O. rhodostigmatus coordinates the capacity of rapid melanogenesis (or other types of pigment production) and pigment regression, a couple of seemingly contradictory coloration requirements. To our knowledge, this is the first study reporting the association between pigmentation phenotype adaptation and MC1R mutations in amphibians or in non-obligative cave dwellers. Overall design: The light-induced skin darkening in the cave-adapted tadpoles of O. rhodostigmatus was investigated based on comparative transcriptomics. Firstly, genes showed a light-depended expression pattern were screened, and functional enrichment analyses were conducted to present the primary cellular processes in response to light exposure; Secondly, the expression pattern of genes involved in pigmentation were analyzed exclusively to determine the coloration types in O. rhodostigmatus tadpoles; Thirdly, genes showed close correlation with critical effector genes responsible for skin darkening in O. rhodostigmatus tadpoles were screened and a functional enrichment analysis were underwent to highlight the potential upstream regulation pathways; Finally, genes potentially contributing to genetic adaptation of coloration were analyzed by sequence aligning and protein homologous modeling to screen functional evolution responsible for the life history of O. rhodostigmatus. Based on the results, we mainly attempt to answer three questions: