The salinity effect on giant grouper innate immunity, Na+-K+-2Cl-cotransporter (NKCC2) and betanodavirus replication
龍膽石斑在台灣是高經濟海水養殖魚種，魚苗時期易受到神經壞死症病毒(NNV)的侵襲。在先前的研究中發現，受NNV感染後存活的石斑苗，其鈉鉀氯共轉運蛋白2 (NKCC2) 的基因表現量比對照組魚高很多。本研究中，選殖了NKCC2 cDNA，並檢視此基因在不同組織中的表現量，發現NKCC2主要在腸道組織中表現，也微量在腦中表現。在30 ppt 鹽度下飼養的魚比 15 ppt 鹽度下飼養的魚有較高的 NKCC2 基因表現，推測腸道中NKCC2吸收鹽份可幫助魚在高鹽度下調節水份的平衡。至今，鹽度如何影響病原對魚致死率的相關報告仍十分有限，本研究因此探討30 ppt與15 ppt兩種海水鹽度對石斑魚攻毒NNV後的累積死亡率，及對NKCC2與先天免疫相關基因的表現有何影響。實驗結果發現，在30 ppt與15 ppt海水飼養下的石斑魚，經NNV攻毒後，30 ppt組的累積死亡率高於15 ppt 組；將30 ppt組的魚於NNV攻毒後飼養鹽度降至15 ppt則累積死亡率下降；反之，將15 ppt組的魚在NNV攻毒後飼養鹽度升至30 ppt則累積死亡率則上升。分析攻毒後1-5天活魚體內先天免疫基因的表現，發現 Mx, TLR-3, MDA-5及C3等基因的表現量變化皆是先升高後緩降，但發炎反應指標基因IL-1β在頭腎及脾臟的表現量卻在感染後第3-5天持續上升。攻毒後第1天的活魚體內， Mx基因表現量是30 ppt組高於15 ppt組；在攻毒後第3-5天，30 ppt組活魚腦中的NNV RNA2表現量則低於15 ppt組，推測與Mx 的表現量有關。此外，30 ppt組感染NNV後3-5日的白血球數目顯著高於15 ppt組。分析感染後第10天收集死亡一日之內的魚，15 ppt組的NNV RNA2表現量明顯高於30 ppt組，但是30 ppt組的IL-1β基因表現量卻明顯高於15 ppt組。NNVRNA2及IL-1β基因在攻毒後14天收集的殘活魚體內的表現量都遠遠低於急性發病期魚或死魚體內的表現量。目前仍不清楚為何高鹽度可以誘發較高先天免疫基因的表現，但發炎反應相關基因的表現若持續升高，即便 30 ppt 組腦中病毒量低於15 ppt 組，持續較高的發炎反應可能是導致30 ppt 出現較高死亡率的重要因素之一。
Giant grouper (Epinephelus lanceolatus) is an economically important fish species for marine aquaculture in Taiwan, but has been attacked by nervous necrosis virus (NNV) for years. In our previous study, high expression level of Na+-K+-2Cl- cotransporter 2 (NKCC2) was found in the survivor grouper larvae. In this study, the full length cDNA of NKCC2 was cloned, and its highest expression level was detected in the intestine, and low level expression was found in the brain. NKCC2 is a membrane channel protein for NaCl adsorption, and we found that its expression level in 30 ppt-acclimated grouper was higher than that in 15 ppt-acclimated fish. The adsorption of salt in intestine in marine fish is suggested to aid the passive adsorption of water for water homeostasis. To date, the information about the impact of salinity on the pathogen-induced mortality is still limited. Therefore, we compared the expression levels of NKCC2 gene, NNV RNA2 and some innate immunity genes, and the accumulated mortality of groupers reared in 30 and 15 ppt sea water after NNV infection. The cumulated mortality of NNV-challenged groupers reared in 30 ppt sea water was higher than that reared in 15 ppt sea water. When the salinity decreased from 30 ppt to 15 ppt after NNV infection, the cumulated mortality declined from 90% to 80%. On the contrary, as the salinity increased from 15 ppt to 30 ppt post NNV challenge, the cumulated mortality increased from 79% to 91%. During 5 days post infection (dpi), the expression patterns of NKCC2, Mx, TLR-3, MDA-5 and C3 genes were all increased at the first and then smoothly decreased, but the expression level of IL-1β continuously increased at the 5th day in head kidney and spleen. At 1 dpi, Mx gene expression in the fish of 30-ppt group was higher than that in 15 ppt-group; however, NNV RNA2 expression level in 30 ppt-group was lower than that in 15 ppt-group at the following 3rd and 5th days. In the same period of time, the leucocytes count in 30 ppt-group was higher than that of 15 ppt-group. In the fish which died within one day and collected at the 10 dpi, the expression level of NNV RNA2 in 30 ppt-group was lower than that in 15 ppt-group, but the expression level of IL-1β in 30 ppt-group was significantly higher than that of 15 ppt-group. In the survival fish sampled at the 14 dpi, the gene expression levels of NNV RNA2 and IL- 1β were much lower than that in the live and dead fish during acute infection. It is still unclear why higher salinity induced higher expression levels of some innate immune genes, but the inflammation maker gene IL-1β continuously increased at the 5th dpi might be one of the important factors result in higher mortality of the fish reared in 30 ppt salinity.
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