Observation of Black Spot Formation and mitfa Expression Analysis of Misgurnus anguillicaudatus from Poyang Lake

Color pattern plays a vital role in animal survival and communication. The type, distribution, and pigment state of pigment cells, and the reflective ability of iridophores determines body color. It varies adaptively in response to external environmental changes and physiological states. The skin pigmentation pattern reflects the number and arrangement of chromatophores. Some fish with rich color patterns, including egg spot patterns, blotch patterns, melanism, horizontal stripe patterns, and vertical bar patterns, have been studied increasingly. This study observed the formation, distribution, and main pattern of chromatophores in 1–60-day-old Misgurnus anguillicaudatus after hatching. Larval melanocytes were first observed in the yolk sac of loach larvae at 3 h post-hatching. From the larval to juvenile stage at 21 days, larval melanocytes appeared on the loach body surface. From the juvenile stage at 22 days to the adult stage, adult melanocytes appeared on the loach body surface. Iridocytes were first observed in the eyes of one-day-old larvae but not on the body surface until they were 12 days old. Xanthophores appeared on the body surface of seven-day-old juveniles. At 2–21 days post-hatching, the melanocytes in the loaches were larval, and their shape changed from star-to snowflake-shaped before forming a black spot. From 22 days, different morphological adult melanocytes formed on the body surface of the loaches with three types of black spots. Chrysanthemum-shaped melanocytes regularly aggregated into large black spots on large black spot loaches. Round and dendritic melanocytes gathered to form small black spots on small black spot loaches. Dendritic melanocytes were evenly distributed on non-black spot loaches. The pigmentation-related mitfa gene was obtained from M. anguillicaudatus using the rapid amplification of cDNA ends (RACE) approach with the SMARTer RACE 5´/3´ Kit User Manual according to the manufacturer's recommendations and was analyzed using bioinformatics and quantitative methods. The results showed that the mitfa gene encoded a protein with 408 amino acids with a calculated molecular mass of 45.68 kDa and an estimated isoelectric point of 7.16. MITFa contained MITF_TFEB_C_3_N, bHLH-Zip, and DUF 3371 domains. MITFa was well-conserved compared to MITF of various species with a higher degree of sequence similarity with other fishes (58.8%–83.2%). The qRT-PCR results showed that the mitfa mRNA was expressed at all stages of embryonic development and reached a peak value at the fertilization stage. mitfa expression was detected in all examined tissues of the three types of loaches, and the highest level of expression was detected in both muscle and dorsal skin (P < 0.01). This study explored pigmentation formation and mitfa expression, serving as a foundation for gaining further insight into the genetic mechanism of body color formation in M. anguillicaudatus.