低盐胁迫对大底鳉(Fundulus grandis)血浆渗透压、鳃上皮细胞形态和通道蛋白mRNA表达的影响
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摘要
为了研究水环境中的低盐度对大底鳉(Fundulus grandis)的适应性影响,采用生理和荧光定量PCR方法探讨了盐度为5、2、1、0.5和0.1的情况下,不同饲养时间大底鳉的血浆渗透压,鳃上皮细胞超微结构及通道蛋白m RNA表达的变化。饲养时间小于1 d、盐度小于0.5的胁迫可以导致血浆渗透压明显降低(P<0.001);鳃上皮表面泌氯细胞的体积增大、顶隐窝开口扩大或其细胞的形状变为三角形或不规则形。当饲养时间超过3 d时,血浆渗透压以及鳃上皮表面泌氯细胞的形态都恢复到对照组水平(盐度为5);低盐胁迫上调了六种鳃通道蛋白m RNA的表达,并下调了两种鳃通道蛋白m RNA的表达。结果显示:广盐性的大底鳉通过调整鳃上皮的形态及通道蛋白m RNA的表达来维持机体渗透压的平衡状态。
The objective of this paper was to describe the effect of hypoosmotic challenge on the plasma osmolality, ultrastructure of branchial epithelia, and m RNA expression of branchial transporters in Fundulus grandis. Adult fish were reared in the salinity from 5(control) to 2, 1, 0.5 and 0.1, and fish were randomly sampled from each salinity at 6 h, 1 d, 3 d, and 7 d. The results showed that plasma osmolality decreased significantly only in fish transferred to the salinity at 0.5 and 0.1, but recovered to the salinity at 5 control values by 1 d and 3 d, respectively. The ultrastructure of branchial epithelia showed that the surface of apical crypt was larger, and the volume of chloride cell swelled within 6 h in the salinity at 0.5. In salinity at 0.1, chloride cells showed a triangle or irregular shape, and they squeezed surrounding pavement cells. The m RNA expression of six branchial transporters was up-regulated, and the levels of two transporters were down-regulated during hypoosmotic challenges. The regulation of the gill morphology and m RNA expressions of branchial transporters may contribute to the freshwateradaption in Fundulus grandis
The objective of this paper was to describe the effect of hypoosmotic challenge on the plasma osmolality, ultrastructure of branchial epithelia, and m RNA expression of branchial transporters in Fundulus grandis. Adult fish were reared in the salinity from 5(control) to 2, 1, 0.5 and 0.1, and fish were randomly sampled from each salinity at 6 h, 1 d, 3 d, and 7 d. The results showed that plasma osmolality decreased significantly only in fish transferred to the salinity at 0.5 and 0.1, but recovered to the salinity at 5 control values by 1 d and 3 d, respectively. The ultrastructure of branchial epithelia showed that the surface of apical crypt was larger, and the volume of chloride cell swelled within 6 h in the salinity at 0.5. In salinity at 0.1, chloride cells showed a triangle or irregular shape, and they squeezed surrounding pavement cells. The m RNA expression of six branchial transporters was up-regulated, and the levels of two transporters were down-regulated during hypoosmotic challenges. The regulation of the gill morphology and m RNA expressions of branchial transporters may contribute to the freshwateradaption in Fundulus grandis
引文
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[10]MUNNS R,WALLACE P A,TEAKLE N L,et al.Measuring soluble ion concentrations Na+,K+,Cl–in salt-treated plants[J].Methods in Molecular Biology,2010,639(23):371–382.
[11]FLEIGE S,WALF V,HUCH S,et al.Comparison of relative m RNA quantification models and the impact of RNA integrity in quantitative real-time RT-PCR[J].Biotechnology Letters,2006,28(19):1601–1613.
[12]KATOH F,HASEGAWA S,KITA J,et al.Distinct seawater and freshwater types of chloride cells in killifish,Fundulus heteroclitus[J].Canadian Journal of Zoology,2001,79(5):822–829.
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[16]PATTERSON J,BODINIER C,GREEN C.Effects of low salinity media on growth,condition,and gill ion transporter expression in juvenile Gulf killifish,Fundulus grandis[J].Comparative Biochemistry and Physiology Part A:Molecular&Integrative Physiology,2012,161(4):415–421.
[17]KATOH F,KANEKO T.Short-term transformation and long-term replacement of branchial chloride cells in killifish transferred from seawater to freshwater,revealed by morphofunctional observations and a newly established'time-differential double fluorescent staining'technique[J].Journal of Experimental Biology,2003,206(22):4113–4123.
[18]WILSON J M,LAURENT P.Fish gill morphology:inside out[J].Journal of Experimental Zoology,2002,293(3):192–213.
[19]LAURENT P,CHEVALIER C,WOOD C M.Appearance of cuboidal cells in relation to salinity in gills of Fundulus heteroclitus,a species exhibiting branchial Na+but not Cl-uptake in freshwater[J].Cell and Tissue Research,2006,325(3):481–492.
[20]SOLLID J,DE ANGELIS P,GUNDERSEN K,et al.Hypoxia induces adaptive and reversible gross morphological changes in crucian carp gills[J].Journal of Experimental Biology,2003,206(20):3667–3673.
[21]SOLLID J,NILSSON G E.Plasticity of respiratory structures-adaptive remodeling of fish gills induced by ambient oxygen and temperature[J].Respiratory Physiology&Neurobiology,2006,154(1/2):241–251.
[22]TIPSMARK C K,S?RENSEN K J,MADSEN S S.Aquaporin expression dynamics in osmoregulatory tissues of Atlantic salmon during smoltification and seawater acclimation[J].Journal of Experimental Biology,2010,213(3):368–379.
[23]CUTLER C P,CRAMB G.Branchial expression of an aquaporin 3(AQP-3)homologue is downregualted in the European eel(Anguilla anguilla)following seawater acclimation[J].Journal of Experimental Biology,2002,205(17):2643–2651.
[24]VAN ITALIE C M,ANDERSON J M.Claudins and epithelial paracellular transport[J].Annual Review of Physiology,2006,68(1):403–429.
[25]CARLISLE T C,RIBERA A B.Connexin 35b expression in the spinal cord of Danio rerio embryos and larvae[J].Journal of Comparative Neurology,2014,522(4):861–875.
[26]CLELLAND E S,BUI P,BAGHERIE-LACHIDAN M,et al.Spatial and salinity-induced alterations in claudin-3isoform m RNA along the gastrointestinal tract of the pufferfish Tetraodon nigroviridis[J].Comparative Biochemistry and Physiology Part A:Molecular&Integrative Physiology,2010,155(2):154–163.
[27]SAS D,HU M,MOE OW,et al.Effect of claudins 6 and 9on paracellular permeability in MDCK II cells[J].American Journal of Physiology:Regulatory,Integrative and Comparative Physiology,2008,295(5):1713–R1719.
[28]COYNE C B,GAMBLING T M,BOUCHER R C,et al.Role of claudin interactions in airway tight junctional permeability[J].American Journal of Physiology:Lung Cellular and Molecular Physiology,2003,285(5):1166–1178.
[29]SCOTT G R,CLAIBORNE J B,EDWARDS SL,et al.Gene expression after freshwater transfer in gills and opercular epithelia of killifish:insight into divergent mechanisms of ion transport[J].Journal of Experimental Biology,2005,208(14):2719–2729.
[30]TIPSMARK C K,MADSEN S S,BORSKI R J.Effect of salinity on expression of branchial ion transporters in striped bass(Morone saxatilis)[J].Journal of Experimental Zoology Part A:Comparative Experimental Biology,2004,301(12):979–991.
[2]MARSHALL W S.Rapid regulation of Na Cl secretion by estuarine teleost fish:coping strategies for short-duration freshwater exposures[J].Biochimica et Biophysica Acta,2003,1618(2):95–105.
[3]BURNETT K G,BAIN L J,BALDWIN W S,et al.Fundulus as the premier teleost model in environmental biology:Opportunities for new insights using genomics[J].Comparative Biochemistry and Physiology Part D:Genomics Proteomics,2007,2(4):257–286.
[4]EVANS D H,PIERMARINI P M,CHOE K P.The multifunctional fish gill:dominant site of gas exchange,osmoregulation,acid-base regulation,and excretion of nitrogenous waste[J].Physiology Reviews,2005,85(1):97–177.
[5]WHITEHEAD A.The evolutionary radiation of diverse osmotolerant physiologies in Killifish(Fundulus sp.)[J].Evolution,2010,64(7):2070–2085.
[6]WHITEHEAD A,GALVEZ F,ZHANG Shujun,et al.Functional genomics of physiological plasticity and local adaptation in killifish[J].Journal of Heredity,2011,102(5):499–511.
[7]KOLOSOV D,BUI P,CHASIOTIS H,et al.Claudins in teleost fishes[J].Tissue Barriers,2013,1(3):e25391.
[8]WHITEHEAD A,ROACH J L,ZHANG Shujun,et al.Salinity-and population dependent genome regulatory response during osmotic acclimation in the killifish(Fundulus heteroclitus)gill[J].Journal of Experimental Biology,2012,215(8):1293–1305.
[9]BROWN C,GOTHREAUX C,GREEN C.Effects of temperature and salinity during incubation on hatching and yolk utilization of Gulf killifish Fundulus grandis embryos[J].Aquaculture,2011,315(3):335–339.
[10]MUNNS R,WALLACE P A,TEAKLE N L,et al.Measuring soluble ion concentrations Na+,K+,Cl–in salt-treated plants[J].Methods in Molecular Biology,2010,639(23):371–382.
[11]FLEIGE S,WALF V,HUCH S,et al.Comparison of relative m RNA quantification models and the impact of RNA integrity in quantitative real-time RT-PCR[J].Biotechnology Letters,2006,28(19):1601–1613.
[12]KATOH F,HASEGAWA S,KITA J,et al.Distinct seawater and freshwater types of chloride cells in killifish,Fundulus heteroclitus[J].Canadian Journal of Zoology,2001,79(5):822–829.
[13]ALTINOK I,CHAPMAN F A,GALLI S M.Ionic and osmotic regulation capabilities of juvenile Gulf of Mexico sturgeon,Acipenser oxyrinchus de sotoi[J].Comparative Biochemistry and Physiology Part A:Molecular&Integrative Physiology,1998,120(4):609–616.
[14]GAVIN J P,GREG I J.The effect of salinity on growth and survival of juvenile black bream(Acanthopagrus butcheri)[J].Aquaculture,2002,210(1):219–230.
[15]KOZAK G M,BRENNAN R S,BERDAN E L,et al.Functional and population genomic divergence within and between two species of killifish adapted to different osmotic niches[J].Evolution,2014,68(1):63–80.
[16]PATTERSON J,BODINIER C,GREEN C.Effects of low salinity media on growth,condition,and gill ion transporter expression in juvenile Gulf killifish,Fundulus grandis[J].Comparative Biochemistry and Physiology Part A:Molecular&Integrative Physiology,2012,161(4):415–421.
[17]KATOH F,KANEKO T.Short-term transformation and long-term replacement of branchial chloride cells in killifish transferred from seawater to freshwater,revealed by morphofunctional observations and a newly established'time-differential double fluorescent staining'technique[J].Journal of Experimental Biology,2003,206(22):4113–4123.
[18]WILSON J M,LAURENT P.Fish gill morphology:inside out[J].Journal of Experimental Zoology,2002,293(3):192–213.
[19]LAURENT P,CHEVALIER C,WOOD C M.Appearance of cuboidal cells in relation to salinity in gills of Fundulus heteroclitus,a species exhibiting branchial Na+but not Cl-uptake in freshwater[J].Cell and Tissue Research,2006,325(3):481–492.
[20]SOLLID J,DE ANGELIS P,GUNDERSEN K,et al.Hypoxia induces adaptive and reversible gross morphological changes in crucian carp gills[J].Journal of Experimental Biology,2003,206(20):3667–3673.
[21]SOLLID J,NILSSON G E.Plasticity of respiratory structures-adaptive remodeling of fish gills induced by ambient oxygen and temperature[J].Respiratory Physiology&Neurobiology,2006,154(1/2):241–251.
[22]TIPSMARK C K,S?RENSEN K J,MADSEN S S.Aquaporin expression dynamics in osmoregulatory tissues of Atlantic salmon during smoltification and seawater acclimation[J].Journal of Experimental Biology,2010,213(3):368–379.
[23]CUTLER C P,CRAMB G.Branchial expression of an aquaporin 3(AQP-3)homologue is downregualted in the European eel(Anguilla anguilla)following seawater acclimation[J].Journal of Experimental Biology,2002,205(17):2643–2651.
[24]VAN ITALIE C M,ANDERSON J M.Claudins and epithelial paracellular transport[J].Annual Review of Physiology,2006,68(1):403–429.
[25]CARLISLE T C,RIBERA A B.Connexin 35b expression in the spinal cord of Danio rerio embryos and larvae[J].Journal of Comparative Neurology,2014,522(4):861–875.
[26]CLELLAND E S,BUI P,BAGHERIE-LACHIDAN M,et al.Spatial and salinity-induced alterations in claudin-3isoform m RNA along the gastrointestinal tract of the pufferfish Tetraodon nigroviridis[J].Comparative Biochemistry and Physiology Part A:Molecular&Integrative Physiology,2010,155(2):154–163.
[27]SAS D,HU M,MOE OW,et al.Effect of claudins 6 and 9on paracellular permeability in MDCK II cells[J].American Journal of Physiology:Regulatory,Integrative and Comparative Physiology,2008,295(5):1713–R1719.
[28]COYNE C B,GAMBLING T M,BOUCHER R C,et al.Role of claudin interactions in airway tight junctional permeability[J].American Journal of Physiology:Lung Cellular and Molecular Physiology,2003,285(5):1166–1178.
[29]SCOTT G R,CLAIBORNE J B,EDWARDS SL,et al.Gene expression after freshwater transfer in gills and opercular epithelia of killifish:insight into divergent mechanisms of ion transport[J].Journal of Experimental Biology,2005,208(14):2719–2729.
[30]TIPSMARK C K,MADSEN S S,BORSKI R J.Effect of salinity on expression of branchial ion transporters in striped bass(Morone saxatilis)[J].Journal of Experimental Zoology Part A:Comparative Experimental Biology,2004,301(12):979–991.