Pathways Of HCO₃^- Metabolism And Transport In Oreochromis Niloticus Under Carbonate Alkalinities

In addition to maintaining osmotic regulation,the fish also facing the pressure on adjustment of acid-base balance under carbonate-type water environment.It is generally believed that the carbonate alkaline water affects the survival of fish by interfering with its blood buffer system.Among them,HCO₃^-is a composition of the most important buffer system in the blood.Oreochromis niloticus is one of the important freshwater aquaculture fish,which can regulate the accumulation of HCO₃^-through complex regulatory mechanisms to adapt to carbonate alkalinity changes.Under carbonate alkalinity stress experiments of transferring O.niloticus from fresh water to 2 and 4 g/L NaHCO3 water environments,analyzing systematicly in physiological?serum Na+,K+,Cl-,HCO₃^-concentration and serum osmolality?,biochemical?CA?,CA?,SLC4A4 and SLC26A6 enzyme activity?,cellular?types,numbers and size of ionocytes?and molecular?CA ?,CA ?,SLC4A4 and SLC26A6 gene and protein expression?levels.Which is to investigate how tilapia could regulate HCO₃^-under carbonate alkalinity stress,main results are as follow:?1?This paper studied the effects of different alkalinity?0,2 and 4 g/L NaHCO3?stress on morphology of gill ionocytes in O.niloticus by the method of scanning electron micrograph,meanwhile immunohistochemistry of carbonic anhydrase?CA ?,CA ??,Na+/HCO₃^-cotransporter?SLC4A4?,Cl-/HCO₃^-exchanger?SLC26A6?in gill,kidney and intestine were also observed.The surface scanning of the gills showed that ionocytes cells were distributed close to the inter-lamellar regions of gill filaments.According to the apical shapes and size,the ionocytes cells could be divided into four subtypes,subtype ?,subtype ?,subtype ? and subtype ?.The apical size of each subtype of ionocytes cells were positively correlated with the carbonate alkalinity stress strength,and the size of subtype ? cells was changed most obviously?P<0.01?;The ionocytes cells number increased significantly with the increase of carbonate alkalinity stress strength,the number of subtype ? cells was increased most significantly?P < 0.01?.The results of immunohistochemistry showed that CA?,CA?,SLC4A4 and SLC26A6 were expressed in gill and kidney of O.niloticus at freshwater and carbonate alkalinity water.And with the increase of carbonate alkalinity concentration,the positive reactions became stronger.However,no positive reaction was detected in intestinal at fresh water and carbonate alkalinity water.This result suggested that O.niloticus could change the quantity and morphological structure of ionocytes in adaption to carbonate alkaline environment,gill and kidney may play a key role in alkalinity regulation.?2?To understand the osmoregulation of fishes in adaptation to carbonate alkalinity water,O.niloticus were divided into 2,4 g/L NaHCO3 alkaline water from freshwater,and the acute changes of serum osmolality,serum Na+,K+,Cl-,HCO₃^-concentration and ion transport enzymes activity of carbonic anhydrase?CA?,CA??,Na+/HCO₃^-cotransporter?SLC4A4?,Cl-/HCO₃^-exchanger?SLC26A6?in gill,kidney and intestine of different treatments were observed at 0,3,6,12,24,48,72,96 and 192 h posttransfer,respectively.Results showed that the changes of serum osmolality,serum Na+,K+,Cl-,HCO₃^-concentration and activities of ion transport enzymes of O.niloticus were relevant to the carbonate alkalinity stress levels.Meantime,serum osmolality,ion concentration shared a similar uppeak-down change trend,and reached the peak at 24 h.The CA?,CA?,SLC4A4,SLC26A6 activity in gill,kidney and intestine decreased at the beginning,and then increased and reached maximum,finally it decreased again and stabilized.These results showed O.niloticus could adapt to carbon alkaline water to a certain degree,and CA?,CA?,SLC4A4 and SLC26A6 were involved in ion transportation and osmolality balance under carbonate alkalinity stress.?3?In order to investigate the mechanism of HCO₃^-metabolism and transport of carbonate alkalinity to fish,O.niloticus were directly transferred into carbonate alkalinity treatments?2,4 g/L NaHCO3?from freshwater for acute stress.The serum pH,relative expression of genes and proteins of carbonic anhydrase?CA?,CA??,Na+/HCO₃^-cotransporter?SLC4A4?and Cl-/HCO₃^-exchanger?SLC26A6?in gill,kidney and intestine were determined using pH meter,quantitative real-time PCR and Western-blotting post-transfer at 0,3,6,12,24,48,72,96 and 192 h,respectively.The results showed that the serum pH showed “up-peak-down” change trend in all treatment groups.It is speculated that HCO₃^-in the water environment enters the blood after entering the hypertonic environment,which causes the changes of the acid-base balance,which leads to the increase of the blood pH.Quantitative real-time PCR showed that with the rise of carbonate alkalinity value,the overall expression of these genes in gill,kidney and intestine are up-regulated compared with the freshwater control?P<0.05?.Compared with the gill and kidney,the expression of relative HCO₃^-transporters genes in intestine were lower.Western blot results showed that the four proteins were expressed in gill and kidney and decreased at the beginning,then increased and reached maximum,finally decreased again and stabilized.But protein expression wasn't detected in the intestine.These indicated that O.niloticus can regulate the HCO₃^-concentration by starting related genes and proteins in gill,kidney and intestine to maintain the acid-base balance in the carbonate base environment.