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
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摘要
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 excretion is inhibited when fish are acutely exposed to alkaline water. Przewalskii's naked carp, also known as Gymnocypris 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 decreasing by 10 cm per year and the salinity and alkalinity levels are increasing by 7% and 0.5% per year, respectively. Some studies showed that G. przewalskii had evolved a variety of mechanisms, such as osmoregulation and ion regulation with low energy consumption, regulation of HCO_3~– secretion in intestines, and compensatory carbonic 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 alkaline water reduced ammonia excretion but increased urea excretion. Ammonia excretion rate decreased significantly 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 NH_3 to NH_4~+. Consequently, at high pH water caused by high carbonate alkalinity, the partial pressure of NH_3(PNH_3) is predicted to rise in water adjacent to the gill, thus reducing the PNH_3 gradient that drives NH_3 diffusion. However, in the 64 mmol/L group, the ammonia 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 NH_3 partial pressure gradient under high carbonate alkalinity. Urea excretion rate increased significantly after 12–16 h and 20–24 h in 32 mmol/L carbonate alkalinity water and 16–48 h in 64 mmol/L carbonate alkalinity water. The real-time PCR results showed that Rhbg, Rhcg2, and Ut genes were up-regulated under carbonate alkalinity stress. The expression of Rhbg in gills was significantly 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 nitrogen 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 environments. We speculate that the special mechanism of nitrogenous waste excretion facilitate the adaptation of G. przewalskii 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.
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 excretion is inhibited when fish are acutely exposed to alkaline water. Przewalskii's naked carp, also known as Gymnocypris 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 decreasing by 10 cm per year and the salinity and alkalinity levels are increasing by 7% and 0.5% per year, respectively. Some studies showed that G. przewalskii had evolved a variety of mechanisms, such as osmoregulation and ion regulation with low energy consumption, regulation of HCO_3~– secretion in intestines, and compensatory carbonic 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 alkaline water reduced ammonia excretion but increased urea excretion. Ammonia excretion rate decreased significantly 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 NH_3 to NH_4~+. Consequently, at high pH water caused by high carbonate alkalinity, the partial pressure of NH_3(PNH_3) is predicted to rise in water adjacent to the gill, thus reducing the PNH_3 gradient that drives NH_3 diffusion. However, in the 64 mmol/L group, the ammonia 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 NH_3 partial pressure gradient under high carbonate alkalinity. Urea excretion rate increased significantly after 12–16 h and 20–24 h in 32 mmol/L carbonate alkalinity water and 16–48 h in 64 mmol/L carbonate alkalinity water. The real-time PCR results showed that Rhbg, Rhcg2, and Ut genes were up-regulated under carbonate alkalinity stress. The expression of Rhbg in gills was significantly 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 nitrogen 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 environments. We speculate that the special mechanism of nitrogenous waste excretion facilitate the adaptation of G. przewalskii 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.
引文
[1]Shi J Q.Study on current situation and countermeasures of resource protection of Qinghai Lake naked carp[J].Qinghai Science and Technology,2008,15(5):13-16.[史建全.青海湖裸鲤研究现状与资源保护对策[J].青海科技,2008,15(5):13-16.]
[2]Yao Z L,Guo W F,Lai Q F,et al.Gymnocypris przewalskii,decreases cytosolic carbonic anhydrase expression to compensate for respiratory alkalosis and osmoregulation in the saline-alkaline lake Qinghai[J].J Comp Physiol B,2016,186(1):83-95.
[3]Shi J Q,Qi H F,Yang J X,et al.Resource Monitoring and Artificial Freshwater Aquaculture Technology of Qinghai Lake Naked Carp[M].Xining:Qinghai Nationalities Publishing House,2012:75-84.[史建全,祁洪芳,杨建新,等.青海湖裸鲤资源监测与淡水全人工养殖技术[M].西宁:青海民族出版社,2012:75-84.]
[4]Wilson J M,Iwata K,Iwama G K,et al.Inhibition of ammonia excretion and production in rainbow trout during severe alkaline exposure[J].Comp Biochem Phys B,1998,121(1):99-109.
[5]Liu J Y,Yao Z L,Lai Q F,et al.Effects of saline-alkali stress on the oxygen consumption and plasma osmolality and ion concentrations of Gymnocypris przewalski[J].Chinese Journal of Ecology,2012,31(3):664-669.[刘济源,么宗利,来琦芳,等.盐碱胁迫对青海湖裸鲤耗氧率、血浆渗透浓度和离子浓度的影响[J].生态学杂志,2012,31(3):664-669.]
[6]Wang Z,Yao Z L,Lin T T,et al.Effects of carbonate alkalinity stress on SOD,ACP,and AKP activities in the liver and kidney of juvenile Gymnocypris przewalskii[J].Journal of Fishery Sciences of China,2013,20(6):1212-1218.[王卓,么宗利,林听听,等.碳酸盐碱度对青海湖裸鲤幼鱼肝和肾SOD、ACP和AKP酶活性的影响[J].中国水产科学,2013,20(6):1212-1218.]
[7]Wang P,Lai Q F,Yao Z L,et al.Differential expressions of genes related to HCO3-secretion in the intestine of Gymnocypris przewalskiii during saline-alkaline water transfer[J].Marine Fisheries,2015,37(4):341-348.[王萍,来琦芳,么宗利,等.盐碱环境下青海湖裸鲤肠道HCO3-分泌相关基因表达差异[J].海洋渔业,2015,37(4):341-348.]
[8]Weihrauch D,Wilkie M P,Walsh P J.Ammonia and urea transporters in gills of fish and aquatic crustaceans[J].J Exp Biol,2009,212(11):1716-1730.
[9]Evans D H,Cameron J N.Gill ammonia transport[J].J Exp Zool,1986,239(1):17-23.
[10]Wood C M.Ammonia and Urea Metabolism and Excretion[M]//Evans D H(eds).The Physiology of Fishes.Boca Raton:CRC Press.1993,77:1-20.
[11]Wright P A,Wood C M,Wright P A,et al.An analysis of branchial ammonia excretion in the freshwater rainbow trout:effects of environmental p H change and sodium uptake blockade[J].J Exp Biol,1985(1):329-353.
[12]Hung C Y C,Tsui K N T,Wilson J M,et al.Molecular cloning,characterization and tissue distribution of the Rhesus Glycoproteins Rh BG,Rh CG1 and Rh CG2 in the Mangrove killifish Rivulusmar moratus exposed to elevated environmental ammonia levels[J].J Biol Chem,2007,210:2419-2429.
[13]Nakada T,Westhoff C M,Kato A,et al.Ammonia secretion from fish gill depends on a set of Rh glycoproteins[J].FASEB J,2007,21(4):1067-1074.
[14]Nawata C M,Hung C C,Tsui T K,et al.Ammonia excretion in rainbow trout(Oncorhynchus mykiss):evidence for Rh glycoprotein and H+-ATPase involvement[J].Physiol Genomics,2007,31(3):463-474.
[15]Wright P A,Wood C M.A new paradigm for ammonia excretion in aquatic animals:role of Rhesus(Rh)glycoproteins[J].J Exp Biol,2009,212(15):2303-2312.
[16]Braun M H,Perry S F.Ammonia and urea excretion in the Pacific hagfish Eptatretus stoutii:evidence for the involvement of Rh and UT proteins[J].Com Biochem Phys A,2010,157(4):405-415.
[17]Wood C M,Nawata C M,Wilson J M,et al.Rh proteins and NH4+-activated Na+-ATPase in the Magadi tilapia(Alcolapia grahami),a 100%ureotelic teleost fish[J].J Exp Biol,2013,216(16):2998-3007.
[18]Kumai Y,Harris J,Alrewashdy H,et al.Nitrogenous waste handling by larval zebrafish Danio rerio in alkaline water[J].Physiol Biochem Zool,2015,88(2):137-145.
[19]He Q,Chang Y M,Su B F,et al.Effects of carbonate alkalinities on oxygen consumption,ammonia excretion and ammonia excretion gene expression in Leuciscus waleckii Dybowski[J].Journal of Shanghai Ocean University,2016,25(4):551-558.[何强,常玉梅,苏宝锋,等.碳酸盐碱度对达里湖瓦氏雅罗鱼耗氧率、氨氮排泄和排氨基因表达的影响[J].上海海洋大学学报,2016,25(4):551-558.]
[20]Wang Y S,Gonzalez R J,Patrick M L,et al.Unusual physiology of scale-less carp,Gymnocypris przewalskii,in Lake Qinghai:a high altitude alkaline saline lake[J].Com Biochem Phys A,2003,134(2):409-421.
[21]Wilkie M P,Wood C M.The adaptations of fish to extremely alkaline environments[J].Com Biochem Phys B,1996,113(4):665-673.
[22]Wilkie M P,Wood C M.Recovery from high p H exposure in the rainbow trout:white muscle ammonia storage,ammonia washout,and the restoration of blood chemistry[J].Physiol Zool,1995,68(3):379-401.
[23]Liu J Y.Effect of saline-alkali stress on oxygen consumption,osmoregulation and ionic regulation of przewalski’s naked carp,Gymnocypris przewalskii[D].Shanghai:Shanghai Ocean University,2012.[刘济源.盐碱胁迫对青海湖裸鲤呼吸耗氧、渗透和离子调节的影响[D].上海:上海海洋大学,2012.]
[24]Lei Y Z.Water Chemistry in Freshwater Aquaculture[M].Nanning:Guangxi Science and Technology Publishing House,1993:65-7,114-116,164-166.[雷衍之.淡水养殖水化学[M].南宁:广西科学技术出版社,1993:65-67,114-116,164-166.]
[25]Verdouw H,Echteld C J A V,Dekkers E M J,et al.Ammonia determination based on indophenol formation with sodium salicylate[J].Water Res,1978,12(6):399-402.
[26]Rahmatullah M,Boyde T R C.Improvements in the determination of urea using diacetyl monoxime;methods with and without deproteinisation[J].Clin Chim Acta,1980,107(1-2):3-9.
[27]Schmittgen T D,Livak K J.Analyzing real-time PCR data by the comparative C(T)method[J].Nat Protoc,2008,3(6):1101-1108.
[28]Salama A,Morgan I J,Wood C M.The linkage between Na+uptake and ammonia excretion in rainbow trout:kinetic analysis,the effects of(NH4)2SO4 and NH4HCO3 infusion and the influence of gill boundary layer p H[J].J Exp Biol,1999,202(6):697-709.
[29]Yesaki T Y,Iwama G K.Survival,acid-base regulation,ion regulation,and ammonia excretion in rainbow trout in highly alkaline hard water[J].Physiol Zool,1992,65(4):763-787.
[30]Thomsen J,Himmerkus N,Holland N,et al.Ammonia excretion in mytilid mussels is facilitated by ciliary beating[J].J Exp Biol,2016,219(15):2300-2310.
[2]Yao Z L,Guo W F,Lai Q F,et al.Gymnocypris przewalskii,decreases cytosolic carbonic anhydrase expression to compensate for respiratory alkalosis and osmoregulation in the saline-alkaline lake Qinghai[J].J Comp Physiol B,2016,186(1):83-95.
[3]Shi J Q,Qi H F,Yang J X,et al.Resource Monitoring and Artificial Freshwater Aquaculture Technology of Qinghai Lake Naked Carp[M].Xining:Qinghai Nationalities Publishing House,2012:75-84.[史建全,祁洪芳,杨建新,等.青海湖裸鲤资源监测与淡水全人工养殖技术[M].西宁:青海民族出版社,2012:75-84.]
[4]Wilson J M,Iwata K,Iwama G K,et al.Inhibition of ammonia excretion and production in rainbow trout during severe alkaline exposure[J].Comp Biochem Phys B,1998,121(1):99-109.
[5]Liu J Y,Yao Z L,Lai Q F,et al.Effects of saline-alkali stress on the oxygen consumption and plasma osmolality and ion concentrations of Gymnocypris przewalski[J].Chinese Journal of Ecology,2012,31(3):664-669.[刘济源,么宗利,来琦芳,等.盐碱胁迫对青海湖裸鲤耗氧率、血浆渗透浓度和离子浓度的影响[J].生态学杂志,2012,31(3):664-669.]
[6]Wang Z,Yao Z L,Lin T T,et al.Effects of carbonate alkalinity stress on SOD,ACP,and AKP activities in the liver and kidney of juvenile Gymnocypris przewalskii[J].Journal of Fishery Sciences of China,2013,20(6):1212-1218.[王卓,么宗利,林听听,等.碳酸盐碱度对青海湖裸鲤幼鱼肝和肾SOD、ACP和AKP酶活性的影响[J].中国水产科学,2013,20(6):1212-1218.]
[7]Wang P,Lai Q F,Yao Z L,et al.Differential expressions of genes related to HCO3-secretion in the intestine of Gymnocypris przewalskiii during saline-alkaline water transfer[J].Marine Fisheries,2015,37(4):341-348.[王萍,来琦芳,么宗利,等.盐碱环境下青海湖裸鲤肠道HCO3-分泌相关基因表达差异[J].海洋渔业,2015,37(4):341-348.]
[8]Weihrauch D,Wilkie M P,Walsh P J.Ammonia and urea transporters in gills of fish and aquatic crustaceans[J].J Exp Biol,2009,212(11):1716-1730.
[9]Evans D H,Cameron J N.Gill ammonia transport[J].J Exp Zool,1986,239(1):17-23.
[10]Wood C M.Ammonia and Urea Metabolism and Excretion[M]//Evans D H(eds).The Physiology of Fishes.Boca Raton:CRC Press.1993,77:1-20.
[11]Wright P A,Wood C M,Wright P A,et al.An analysis of branchial ammonia excretion in the freshwater rainbow trout:effects of environmental p H change and sodium uptake blockade[J].J Exp Biol,1985(1):329-353.
[12]Hung C Y C,Tsui K N T,Wilson J M,et al.Molecular cloning,characterization and tissue distribution of the Rhesus Glycoproteins Rh BG,Rh CG1 and Rh CG2 in the Mangrove killifish Rivulusmar moratus exposed to elevated environmental ammonia levels[J].J Biol Chem,2007,210:2419-2429.
[13]Nakada T,Westhoff C M,Kato A,et al.Ammonia secretion from fish gill depends on a set of Rh glycoproteins[J].FASEB J,2007,21(4):1067-1074.
[14]Nawata C M,Hung C C,Tsui T K,et al.Ammonia excretion in rainbow trout(Oncorhynchus mykiss):evidence for Rh glycoprotein and H+-ATPase involvement[J].Physiol Genomics,2007,31(3):463-474.
[15]Wright P A,Wood C M.A new paradigm for ammonia excretion in aquatic animals:role of Rhesus(Rh)glycoproteins[J].J Exp Biol,2009,212(15):2303-2312.
[16]Braun M H,Perry S F.Ammonia and urea excretion in the Pacific hagfish Eptatretus stoutii:evidence for the involvement of Rh and UT proteins[J].Com Biochem Phys A,2010,157(4):405-415.
[17]Wood C M,Nawata C M,Wilson J M,et al.Rh proteins and NH4+-activated Na+-ATPase in the Magadi tilapia(Alcolapia grahami),a 100%ureotelic teleost fish[J].J Exp Biol,2013,216(16):2998-3007.
[18]Kumai Y,Harris J,Alrewashdy H,et al.Nitrogenous waste handling by larval zebrafish Danio rerio in alkaline water[J].Physiol Biochem Zool,2015,88(2):137-145.
[19]He Q,Chang Y M,Su B F,et al.Effects of carbonate alkalinities on oxygen consumption,ammonia excretion and ammonia excretion gene expression in Leuciscus waleckii Dybowski[J].Journal of Shanghai Ocean University,2016,25(4):551-558.[何强,常玉梅,苏宝锋,等.碳酸盐碱度对达里湖瓦氏雅罗鱼耗氧率、氨氮排泄和排氨基因表达的影响[J].上海海洋大学学报,2016,25(4):551-558.]
[20]Wang Y S,Gonzalez R J,Patrick M L,et al.Unusual physiology of scale-less carp,Gymnocypris przewalskii,in Lake Qinghai:a high altitude alkaline saline lake[J].Com Biochem Phys A,2003,134(2):409-421.
[21]Wilkie M P,Wood C M.The adaptations of fish to extremely alkaline environments[J].Com Biochem Phys B,1996,113(4):665-673.
[22]Wilkie M P,Wood C M.Recovery from high p H exposure in the rainbow trout:white muscle ammonia storage,ammonia washout,and the restoration of blood chemistry[J].Physiol Zool,1995,68(3):379-401.
[23]Liu J Y.Effect of saline-alkali stress on oxygen consumption,osmoregulation and ionic regulation of przewalski’s naked carp,Gymnocypris przewalskii[D].Shanghai:Shanghai Ocean University,2012.[刘济源.盐碱胁迫对青海湖裸鲤呼吸耗氧、渗透和离子调节的影响[D].上海:上海海洋大学,2012.]
[24]Lei Y Z.Water Chemistry in Freshwater Aquaculture[M].Nanning:Guangxi Science and Technology Publishing House,1993:65-7,114-116,164-166.[雷衍之.淡水养殖水化学[M].南宁:广西科学技术出版社,1993:65-67,114-116,164-166.]
[25]Verdouw H,Echteld C J A V,Dekkers E M J,et al.Ammonia determination based on indophenol formation with sodium salicylate[J].Water Res,1978,12(6):399-402.
[26]Rahmatullah M,Boyde T R C.Improvements in the determination of urea using diacetyl monoxime;methods with and without deproteinisation[J].Clin Chim Acta,1980,107(1-2):3-9.
[27]Schmittgen T D,Livak K J.Analyzing real-time PCR data by the comparative C(T)method[J].Nat Protoc,2008,3(6):1101-1108.
[28]Salama A,Morgan I J,Wood C M.The linkage between Na+uptake and ammonia excretion in rainbow trout:kinetic analysis,the effects of(NH4)2SO4 and NH4HCO3 infusion and the influence of gill boundary layer p H[J].J Exp Biol,1999,202(6):697-709.
[29]Yesaki T Y,Iwama G K.Survival,acid-base regulation,ion regulation,and ammonia excretion in rainbow trout in highly alkaline hard water[J].Physiol Zool,1992,65(4):763-787.
[30]Thomsen J,Himmerkus N,Holland N,et al.Ammonia excretion in mytilid mussels is facilitated by ciliary beating[J].J Exp Biol,2016,219(15):2300-2310.