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Molecular characterization of three ion transporter genes from the gills of the giant mudskjpper, Periophthalmodon schlosseri and effects of ammonia exposure or salinity acclimation on their mRNA expression levels
Author
Kang, Charlene Minzhen
Supervisor
Chew, Shit Fun
Abstract
The giant mudskipper, Periophthalmodon schlosseri, is an amphibious fish found living on the mudflats. It has evolved osmoregulatory mechanisms to survive in low and high salinities and is able to actively excrete amn1onia during terrestrial exposure. Both osmoregulation and active excretion of NH4+ may involve Na+:K+:2C1- (Nkcc) present on the basolateral men1brane of the gill epithelium, cystic fibrosis transmembrane conductance regulator ( Cftr ), and vacuolar H+ ATPase ( Atp6v1) present on the apical membrane of the gills. This study was undertaken to elucidate if there exist different isoforms of cftr, nkcc and atp6v1b (Atp6v1 B subunit) in the gills of P. schlosseri and to obtain their full cDNA sequences. The roles of Cftr, Nkcc and Atp6v1 in both osmoregulation and active ammonia excretion were also examined by measuring the mRNA expression of the cftr, nkcc and atp6v1 isoforms in the gills of P. schlosseri after exposure to salinity 5 (low salinity), salinity 5 plus 75 mM NH4C1, salinity 30 (seawater) and salinity 30 plus 75 mM NH4Cl tor 1 d and 6 d. Only one isoform of cftr, nkcc and atp6v1b have been identified in the gills of P. schlosseri. Phylogenetic analyses conducted showed that these isofonns share the highest amino acid sequence identity with teleosts. There was no significant change in mRNA expression of cftrl in the gills of P. schlosseri on exposure to salinity 30 (high salinity) for 1 d as compared to that of fish exposed to salinity 5 (low salinity) for the same period. However, the mRNA expression of cftr1 increased significantly by about 3.4-fold in the gills after P. schlosseri was exposed to salinity 30 for 6 d as compared to that of fish exposed to salinity 5 for the same period. On exposure to salinity 30 for 1 d, the mRNA expression of nkcc1 in the gills of P. schlosseri increased significantly by 12-fold compared to that exposed to salinity 5 for a similar period. As the mRNA expression of nkcc1 was reduced by 42% after 6 d of exposure to salinity 30 as compared to that exposed to the same salinity for 1 d, thus the increase in mRNA expression of nkcc1 was only 7.9-fold that of fish exposed to salinity 5 for the same period of 6 d. Exposure of P. schlosseri to salinity 30 for 1 d led to a significant decrease in the mRNA expression of atp6v1b1 in the gills as con1pared to that exposed to salinity 5 for the same period. However, a prolonged exposure of 6 d to salinity 30 did not affect the mRNA expression of atp6v1b1 compared to that exposed to salinity 5 for a similar period. The mRNA expression of atp6v1b1 in the gills of fish exposed to 75 mM NH4C1 at salinity 5 after 1 d or 6 d did not increase significantly compared to the corresponding fish exposed to salinity 5 alone. Although the mRNA expression of atp6v1b1 in the gills of P. schlosseri also did not increase significantly after exposure to 75 mM NH4C1 at salinity 30 for 1 d, there was a significant increase after the fish was exposed for 6 d. Thus, this study showed that Cftr and Nkcc are important for high salinity adaptation in P. schlosseri while Atp6v1 is important in maintaining the acid boundary to prevent the backflux of NH4+ (that was excreted) as NH3 during ammonia loading.
Date Issued
2013
Call Number
QL638.G7 Kan
Date Submitted
2013