高碱环境下青海湖裸鲤氮废物排泄及相关基因的表达规律

Nitrogenous waste excretion and gene expression of nitrogen transporter in Gymnocypris przewalskii in high alkaline environment

采用个体生理学及定量PCR方法,研究了青海湖裸鲤(Gymnocypris przewalskii)在32 mmol/L(CA32)和64 mmol/L(CA64)碳酸盐碱度胁迫下氮废物排泄规律及鳃和肾组织中Rhesus type b glycoproteins(Rhbg)、Rhesus type c2 glycoproteins(Rhcg2)与urea transporter(Ut)基因的表达规律。结果显示,在32 mmol/L碱度胁迫整个过程中及64 mmol/L碱度胁迫初期裸鲤氨氮排泄率显著降低,但在64 mmol/L碱度胁迫8~20 h、24~72 h时氨氮排泄率基本恢复到胁迫前水平,在32 mmol/L碱度胁迫12~16 h、20~24 h及64 mmol/L碱度胁迫16~48 h时尿素氮排泄率显著升高。定量PCR结果显示,Rhbg、Rhcg2、Ut基因在胁迫过程中都有表达上调趋势,其中32 mmol/L碱性环境下鳃组织中Rhbg基因在胁迫12 h时表达明显上调;64 mmol/L碱性环境下鳃组织中Rhcg2基因在胁迫6 h、48 h、72 h表达明显上调,Ut基因在胁迫6 h表达明显上调,肾组织中Rhcg2基因在胁迫6 h表达明显上调。以上结果表明,裸鲤在高碱环境下虽然前期氮废物排泄受到抑制,但后期会通过启动Rh基因高表达恢复氨氮排泄,同时启动Ut高表达来增加尿素氮排泄来进行氮废物排泄。这一特殊氮废物排泄策略有助于裸鲤更好地适应高碱性环境。

Saline-alkaline water bodies are widely distributed in China. High alkalinity is one of the main stressors for the survival of aquatic animals in saline-alkaline water. Previous studies have established that ammonia excre- tion is inhibited when fish are acutely exposed to alkaline water. Przewalskii＇s naked carp, also known as Gymno- cypris przewalskii or the scale-less carp, is endemic to the austere environment of Lake Qinghai. Lake Qinghai has a high salinity （13 ppt） and a strong alkalinity （carbonate alkalinity approximately 29 mmol/L, pH 9.1-9.5）. Due to high evaporative water loss and extensive water diversion for agricultural use, the water level of the lake is de- creasing by 10 cm per year and the salinity and alkalinity levels are increasing by 7% and 0.5% per year, respec- tively. Some studies showed that G. przewalskii had evolved a variety of mechanisms, such as osmoregulation and ion regulation with low energy consumption, regulation of HCO3 secretion in intestines, and compensatory car- bonic anhydrase expression mechanism under metabolic alkalosis, to adapt to saline-alkaline environments. However, the mechanism of nitrogenous waste excretion is less well studied. In order to evaluate the effect of carbonate alkalinity stress on nitrogenous waste excretion in G. przewalskii, we exposed juvenile G przewalskii to 32 mmol/L and 64 mmol/L carbonate alkalinity water and measured ammonia and urea excretion rate after -6 h （pre-transfer）, 4 h, 8 h, 12 h, 16 h, 20 h, 24 h, 48 h, 72 h, 96 h, and 120 h （recovery） after initial exposure. We also measured Rhesus type b glycoproteins （Rhbg）, Rhesus type c2 glycoproteins （Rhcg2）, and urea transporter （Ut） expression in gill and kidney of G. przewalskii by real-time PCR. The results showed that G. przewalskii in alka- line water reduced ammonia excretion but increased urea excretion. Ammonia excretion rate decreased signifi- cantly over the entire exposure period in 32 mmol/L carbonate alkalinity water and the initial exposure period in 64 mmol/L carbonate alkalinity water. Ammonia excretion is expected to be inhibited when fish are subjected to alkaline water because of a decrease in the extent of the protonation of NH3 to NH~. Consequently, at high pH water caused by high carbonate alkalinity, the partial pressure of NH3 （PNH3） is predicted to rise in water adjacent to the gill, thus reducing the PNH3 gradient that drives NH3 diffusion. However, in the 64 mmol/L group, the am- monia excretion rate recovered to the level of pre-transfer after 24-72 h and 8-20 h. This indicated that G. przewalskii may excrete ammonia by re：establishing a favorable NH3 partial pressure gradient under high carbon- ate alkalinity. Urea excretioo rate increased significantly after 12-16 h and 20-24 h in 32 mmol/L carbonate alka- linity water and 16-48 h in 64 mmol/L carbonate alkalinity water. The real-time PCR results showed that Rhbg, RtTcg2, and Ut genes were up-regulated under carbonate alkalinity stress. The expression of Rhbg in gills was signifi- cantly up-regulated after 12 h in 32 mmol/L carbonate alkalinity water, while Rhcg2 was significantly up-regulated in gills after 6 h, 48 h, and 72 h and in kidneys at 6 h in 64 mmol/L carbonate alkalinity water. Ut expression in gills was significantly up-regulated after 6 h in the 64 mmol/L carbonate alkalinity group. These results revealed that although ammonia excretion was inhibited in highly alkaline environments, G. przewalskii could excrete ni- trogen waste by up-regulating Rh and Ut expression, recovering ammonia excretion, and excreting more urea. This study provided evidence of the nitrogenous waste excretion mechanism in G. przewalskii in high alkaline envi- ronments. We speculate that the special mechanism of nitrogenous waste excretion facilitate the adaptation of G przewalsldi to highly alkaline environments. Nonetheless, these findings raise more questions than answers, and further studies are needed to clarify the distribution and expression level of Rh protein in cells and tissues.