Artificial insemination techniques and molecular insights into iridophore development in the ornamental fish bitterling (Rhodeus ocellatus)

The body color of fish is a significant external morphological characteristic that often influences both their ornamental and economic value. Iridophores, display a spectrum of colors that varies with the wavelength of reflected light and constitute one of the predominant types of pigment cells involved in fish coloration. Bitterling is a kind of small freshwater ornamental fish. The mature body surface of male bitterling is brilliantly coloured and distributed with abundant iridophores, making it a great material for the study of iridophores development. However, it's difficult to study the body colour of bitterling under laboratory conditions due to the special breeding method, which requires mussels to accomplish fertilization and incubation. Exploring effective artificial insemination techniques for bitterling is the key to overcoming this problem. In this study, the ratio of spawning tube length to body length and body colour characteristics were used as the basis for female and male parental selection of bitterling, respectively, to establish an efficient artificial insemination technique for bitterling., which significantly improved the fertilization rate (96%), hatching rate (92%), and survival rate (80%). During the process of body color development of bitterling, the number of iridophores in the outermost fin of ventral increased continuously, and gradually formed a bright silvery-white color on the body surface. Ultrastructural observation revealed that the iridophores in the ventral fins of sexually mature male bitterling exhibit two distinct morphological structures, long spusiform and round granular. The number, distribution and morphological structure of iridophores play an important role in the body color formation of bitterling. The qPCR expressions of candidate genes pnp , edar , bmp4 , dsg2 and dsc2 at different stages of body color development were compared and analyzed. These genes may be related to the development of the ventral fin of iridophores.The above research results provide a theoretical basis for exploring the molecular regulatory mechanisms of fish body color development and diversity. • An efficient method for identifying the parental individuals of bitterling and an artificial insemination technique have been established. • The color development process of the bitterling was characterized. • A total of 5 candidate functional genes probable related to the development of iridophore cells were identified.