Cave-dwelling animals evolve distinct troglomorphic traits, such as loss of eyes, skin pigmentation, and augmentation of senses following long-term adaptation to the perpetual darkness. However, the molecular genetic mechanisms underlying these phenotypic variations remain unclear. In this study, we conducted comparative histology and comparative transcriptomics study of the skin of eight Sinocyclocheilus species (Cypriniformes: Cyprinidae) that included surface and cave-dwelling species. We analyzed four surface and four cavefish species by using next-generation sequencing, and a total of 802,798,907 clean reads were generated and assembled into 505,495,009 transcripts, which contributed to 1,037,334 unigenes. Bioinformatics comparisons of four different surface-cave fish groups revealed between 10,629 and 6442 significantly differentially expressed unigenes. Further, tens of differentially expressed genes (DEGs) potentially related to skin pigmentation were identified. Most of these DEGs (including GNAQ, PKA, NRAS, and p38) are downregulated in cavefish species. They are involved in key signaling pathways of pigment synthesis, such as the melanogenesis, Wnt, and MAPK pathways. This trend of downregulation was confirmed through qPCR experiments. This study will deepen our understanding of the formation of troglomorphic traits in cavefishes.

Raw data (raw reads) in the fastq format were firstly processed through in-house perl scripts. In this step, clean data (clean reads) were obtained by removing reads containing adapter, reads containing ploy-N, and low-quality reads from raw data. At the same time, Q20, Q30, GC-content, and sequence duplication level of the clean data were calculated. All the downstream analyses were based on clean data with high quality, and then transcriptome assembly was conducted using Trinity (Grabherr et al., 2011), with min_kmer_cov set to 2 by default; all other parameters were set to default values.