建立臺灣常見觀賞魚－印度女王燈（恆河青鱂魚；Oryzias dancena）成為魚類生理學研究的新平台

Abstract

Abstract Brackish medaka (Oryzias dancena) is a famous ornamental fish in the local aquaria. This medaka species is close to Japanese medaka (O. latipes) with genomic database. Individuals which the study uses are obtained from the local aquaculture farms in south Taiwan. Since the brackish medaka was characterized by it’s euryhalinity, easy and economic to rear, stable resources, and available of medaka genomic database (Japanese medaka), the goal of the present study was to develop this fish to be a new experimental species for physiological studies. This thesis attempted to explore the osmoregulatory mechanism and morphology of mitochondrion-rich (MR) cells in gills of brackish medaka upon various environmental salinities. From genetic to cellular expressions of gill Na+, K+-ATPase (NKA) were compared between Japanese medaka and brackish medaka acclimated to fresh water (FW), brackish water (BW), and seawater (SW) to illustrate the conformance to the hypothesis that the lowest NKA activity exhibited in the gills of euryhaline teleosts upon the environments with salinity similar to their nature habitats. On the other hand, expressions of gill Na+, K+, 2Cl- cotransporter 1 (NKCC1), a member of Cl- secretory mechanism, were evaluated in the in the brackish medaka. We found that the Odnkcc1a gene was dominantly found in gill MR cells using whole-mount in situ hybridization. The gene and protein expressions of NKCC1a were both salinity-dependent in gills of brackish medaka. Furthermore, transfer of brackish medaka from SW to FW revealed that protein abundance of NKCC1a in gills was retained until 7 days, which is a likely mechanism for maintaining hyposmoregulatory endurance of this fish. In addition, this study observed time-course remodeling of the cell remodeling and the altered expressions of typical ion transporters in the branchial MR cells of SW-acclimated brackish medaka when exposed to FW. The dynamic elimination of Cl- secretory capacity of branchial MR cells exhibited two phases. In the 1st day post-transfer, the first phase showed rapid changes: the hole-type apical surface of MR cells started to replace the flat-type cells, the apical immunostaining signals of cystic fibrosis transmembrane regulator (CFTR) disappeared, and the gene abundances of Odnkcc1a and Odcftr both decreased. From the second phase (the 2nd day after transfer), the basolateral immunostaining signals of NKCC1a protein were vanishing. At this period, the observed proliferated MR cells no more exhibited the Cl- secretory capacity. The microvilli were commonly found in gill ionocytes of the fresh water- (FW-) adapted fish rather than in those of the seawater- (SW-) adapted fish. Using the brackish medaka, this study characterized villin-like (VILL) protein and illustrated the correlation between apical microvilli of the ionocytes and fish VILL expression. The highest mRNA and protein levels of VILL were found in gills of FW-acclimated medaka compared to the SW and BW fish. Imufluorescence staining combined with vital staining (Rhodamine 123) and scanning electron microscopy revealed that the distribution of VILL protein was in the cell cortex of FW-type MR cells with microvilli. Finally, the gene knockdown technology of morpholino oligonucleotides (MO) was established in the brackish medaka embryo. The VILL-MO results indicated that the VILL protein was involved in the formation of microvilli in the apical surface of FW-type MR cells. To our knowledge, this is the first study to illustrate the morphological function of fish VILL protein. Taken together, different approaches were used in this study to address the mechanisms of fish osmoregulation and develop the brackish medaka to be a new experimental animal. The integrative study will provide new insights in the fish ionoregulatory researches.