| Summary: | 碩士 === 國立中興大學 === 生命科學系所 === 103 === Osmoregulation is the active regulation of the osmotic pressure of an organism’s fluids by behavior or actively transporting ions across cell membrane to maintain the organism’s water content. Gill is the most important osmoregulatory organ for teleosts. Previous studies reported that fish gill possess a specific type of epithelial cell, the ionocyte, which was characterized by a number of mitochondria in the cytoplasm, and was able to actively transport ions across the cell membrane. Medakas represent teleostean species of the genus Oryzias. Two phylogenetic groups of medakas were found including fish exhibiting euryhality, the javanicus species group and latipes species group, and one phylogenetic group of fish exhibiting stenohality, the celebensis species group. Daisy’s medaka, Oryzias woworae was a recently described medaka species, which inhabit in the upstream freshwater (FW) river of Sulawesi island, Indonesia. The present study was aimed to investigate osmoregulatory and cytoprotective mechanisms in gills of the Daisy’s medaka when exposed to osmotic stress. These results will also establish a comparative model fish for studying the difference in gill osmoregulatory mechanisms between euryhaline and stenohaline teleosts.
In the first chapter, phylogeny analysis of medaka by 12S and 16S rDNA revealed that O. woworae was categorized into the celebensis species group. Salinity tolerance test indicated that Daisy’s medaka was a stenohaline teleost. Only 53.3% fish survived after one week transfer from FW to 10‰ salt water (SW). However, pre-acclimated to 5‰ SW for one week increased the survive rate of Daisy’s medaka to 62.5% when transferred to 10‰ SW. Hence, this transfer regime was used in the following experiments. Plasma analysis revealed that plasma osmolality, sodium and chloride concentration significantly increased in response to 10‰ SW. Protein expression and activity of Na+, K+-ATPase (NKA) were found significantly increased in 10‰ SW group. Anti-NKA antibody was used to label ionocytes. Larger size and more number of ionocytes appeared in the gill of 10‰ SW fish. In the 10‰ SW group, protein expression of branchial Na+, K+, 2Cl- cotransporter 1 (NKCC1) were also significantly increased. Confocal microscopic observation revealed that NKCC1 was localized to the basolateral membrane of ionocytes. Moreover, the present study revealed that protein expression of gill NKCC1 was higher in O. dancena (euryhaline medaka) than that in O. woworae (stenohaline medaka) at the same quantity of gill lysates.
In the second chapter, transmission electron microscopic observation revealed higher density volume of mitochondria in the gill ionocyte of 10‰ SW fish. Protein expression of a mitochondrial maker, cytochrome c oxidase subunit IV (COX IV), was also found significantly increased in the gill of 10‰ SW fish. Full-length analysis of heat shock protein 60 (hsp60) sequence revealed conserved functional motifs of HSP60 in the deduced protein structure. Tissue distribution analysis by RT-PCR revealed that hsp60 gene was mainly expressed in the eye, gill, liver and intestine of Daisy’s medaka. Elevation of hsp60 gene expression was confirmed in the gill of Daisy’s medaka in 10‰ SW group by real-time PCR. Protein expression of HSP60 was also found significantly increased in the gill of 10‰ SW fish. Results of double immunofluorecence staining revealed immunoreactivity of HSP60 in the gill ionocyte. In this study, Leibovitz’s L-15 medium, of which osmolality was similar to 10‰ SW, was used as a treatment to the gill of FW Daisy’s medaka in vitro. After in vitro gill culture, gene expression of hsp60 significantly increased compared to the FW fish gill.
Taken together, Daisy’s medaka, which is a stenohaline teleost, would activate osmoregulatory and cytoprotective mechanism in the gill uopn 10‰ SW salinity challenge. In addition, the congener of euryhaline medaka was available to do the comparative studies on osmoregulatory mechanisms between euryhaline and stenohaline species. The present study indicated that Daisy’s medaka would be a good stenohaline model fish.
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