Molecular Basis of Color Variation in Taiwanese Loach Revealed by Early Developmental Transcriptome Analysis

Background: The Taiwanese loach (Paramisgurnus dabryanus ssp. Taiwan, Dabry de Thiersant, 1872.) is an economically important aquaculture species in East Asia, and its body color directly affects its ornamental and market value. Our research group recently discovered a golden-red mutant, named “Gan Hong No. 1” (MR), within a wild-type (WT) population. During embryogenesis, MR individuals exhibit almost no melanophore deposition, and after hatching, xanthophores and erythrophores appear sequentially, suggesting that the body color variation likely originates from alterations in the gene regulatory network during early development. Objective: To systematically compare the transcriptomes of WT and MR-Taiwanese loach during early development, to identify the key regulatory pathways underlying red body color formation from a temporal perspective, to test whether the classical melanin synthesis pathway is impaired, and to provide a theoretical basis for selective breeding of body color traits. Methods: High-throughput transcriptome sequencing was performed on eight early developmental stages (0, 5, 10, 15, 20, 23, 28 and 43 h post-fertilization) of both loach types. Differential expression analysis, time-series trend analysis, and Kyoto encyclopedia of genes and genomes (KEGG) pathway enrichment were used to systematically characterize gene expression dynamics. Transcriptomic data validation was performed using real-time PCR. Results: In MR, the core transcription factor mitfa was significantly downregulated, whereas the expression of melanin synthesis genes such as kita and dct showed no significant difference, indicating that the impairment of melanogenesis is caused by mitfa downregulation. Trend analysis and pathway enrichment revealed that in MR embryos, pathways related to oxidative stress, unsaturated fatty acid biosynthesis, C-type lectin receptor signaling, p53 signaling, and apoptosis were significantly activated, while the thyroid hormone synthesis pathway was markedly upregulated. In WT, these pathways showed the opposite trend. qRT-PCR results were consistent with the transcriptome data. Conclusions: This study demonstrates that downregulation of mitfa serves as the initial trigger for red body color variation in the Taiwanese loach. This mutation impedes melanin synthesis and concurrently activates a coordinated regulatory network involving oxidative stress, immune inflammation, and thyroid hormone signaling. Accumulation of unsaturated fatty acids alleviates oxidative damage and supports carotenoid deposition, while immune signals eliminate aberrant melanocytes and promote compensatory generation of red and yellow chromatophores. The upregulated thyroid hormone further fine-tunes pigment cell differentiation. For the first time in a cobitid species, this study elucidates the mitfa-mediated, multi-pathway synergistic molecular mechanism driving the transition from melanin-based to carotenoid/pteridine-based red coloration in fish, thereby providing a theoretical reference for molecular breeding of body color in aquaculture.