Mechanisms of Ca2+ uptake in freshwater and seawater-acclimated killifish, Fundulus heteroclitus, and their response to acute salinity transfer

  • Alex M. Zimmer
  • Kevin V. Brix
  • Chris M. Wood
Original Paper


Killifish (Fundulus heteroclitus) has been extensively used as a model for ion regulation by euryhaline fishes. Na+ and Cl dynamics have been well studied in killifish, but few studies have addressed that of Ca2+. Therefore, this study aimed to characterize Ca2+ fluxes in freshwater (FW) and seawater (SW)-acclimated killifish, their response to salinity transfer, and to elucidate the mechanisms of Ca2+ influx in FW and SW. SW killifish displayed a significantly higher Ca2+ influx rate than that of FW fish, while Ca2+ efflux rates were comparable in both salinities. Ca2+ influx was saturable in FW (Km = 78 ± 19 µmol/L; Jmax = 53 ± 3 nmol/g/h) and influx by SW killifish was linear up to 7 mmol/L Ca2+. In SW-acclimated fish, 36% of Ca2+ influx was attributed to “intestinal Ca2+ intake”, likely caused by drinking, whereas intestinal Ca2+ intake in FW contributed to < 2% of total. Throughout the study, results suggested that “cation competition” in SW modulates Ca2+ influx. Therefore, we hypothesized that SW-acclimated fish actually have a higher affinity Ca2+ influx system than FW-acclimated fish but that it is competitively inhibited by competing SW cations. In agreement with this cation competition hypothesis, we demonstrated for the first time that “extra-intestinal” Ca2+ influx was inhibited by Mg2+ in both FW and SW-acclimated killifish. Following acute salinity transfer, extra-intestinal Ca2+ influx was rapidly regulated within 12–24 h, similar to Na+ and Cl. Ca2+ influx in FW was inhibited by La3+, an epithelial Ca2+ channel blocker, whereas La3+ had no significant effect in SW.


Ion regulation Salinity Epithelial Ca2+ channel (ECaC) Cation competition Osmoregulation Drinking 



Special thanks are given to Linda Diao who conducted many of the experiments and analyses at McMaster University, to Sunita Nadella who also helped with experiments and analyses, and to Marina Giacomin who assisted with experiments at UBC. Constructive comments from four anonymous reviewers improved the MS. Funded by a NSERC Discovery Grant to CMW. CMW was supported by the Canada Research Chairs Program. KVB was supported by a NSF Post-Doctoral Fellowship (DBI-1306452) and by a NSERC Discovery Grant to CMW. AMZ is supported by a NSERC Post-doctoral fellowship.


  1. Baldisserotto B, Chowdhury MJ, Wood CM (2005) Effects of dietary calcium and cadmium on cadmium accumulation, calcium and cadmium uptake from the water, and their interactions in juvenile rainbow trout. Aquat Toxicol 72:99–117CrossRefPubMedGoogle Scholar
  2. Blewett T, MacLatchy DL, Wood CM (2013) The effects of temperature and salinity on 17-α-ethynylestradiol uptake and its relationship to oxygen consumption in the model euryhaline teleost (Fundulus heteroclitus). Aquat Toxicol 127:61–71CrossRefPubMedGoogle Scholar
  3. Burnett KG, Bain LJ, Baldwin WS, Callard GV, Cohen S, Di Giulio RT, Evans DH, Gómez-Chiarri M, Hahn ME, Hoover CA, Karchner SI, Katoh F, MacLatchy DL, Marshall WS, Meyer JN, Nacci DE, Oleksiak MF, Rees BB, Singer TD, Stegeman JJ, Towle DW, Van Veld PA, Vogelbein WK, Whitehead A, Winn RN, Crawford DL (2007) Fundulus as the premier teleost model in environmental biology: opportunities for new insights using genomics. Comp Biochem Physiol D 2:257–286Google Scholar
  4. Dymowska AK, Hwang PP, Goss GG (2012) Structure and function of ionocytes in the freshwater fish gill. Respir Physiol Neurobiol 184:282–292CrossRefPubMedGoogle Scholar
  5. Evans DH, Piermarini PM, Choe KP (2005) The multifunctional fish gill: dominant site of gas exchange, osmoregulation, acid-base regulation, and excretion of nitrogenous waste. Physiol Rev 85:97–177CrossRefPubMedGoogle Scholar
  6. Flik G, Wendelaar Bonga SE, Fenwick JC (1983) Ca2+-dependent phosphatase and ATPase activities in eel gill plasma membranes-I. Identification of Ca2+-activated ATPase activities with non-specific phosphatase activities. Comp Biochem Physiol B 76:745–754CrossRefPubMedGoogle Scholar
  7. Flik G, van Rijs JH, Wendelaar Bonga SE (1985a) Evidence for high-affinity Ca2+-ATPase activity and ATP-driven Ca2+-transport in membrane preparations of the gill epithelium of the cichlid fish Oreochromis mossambicus. J Exp Biol 119:335–347Google Scholar
  8. Flik G, Wendelaar Bonga SE, Fenwick JC (1985b) Active Ca2+ transport in plasma membranes of branchial epithelium of the North-American eel, Anguilla rostrata LeSueur. Biol Cell 55:265–272CrossRefPubMedGoogle Scholar
  9. Flik G, Van Der Velden JA, Dechering KJ, Verbost PM, Schoenmakers TJM, Kolar ZI, Bonga SEW (1993) Ca2+ and Mg2+ transport in gills and gut of tilapia, Oreochromis mossambicus: a review. J Exp Zool 265:356–365CrossRefGoogle Scholar
  10. Flik G, Klaren PHM, Schoenmakers TJM, Bijvelds MJC, Verbost PM, Wendelaar Bonga SE (1996) Cellular calcium transport in fish: unique and universal mechanisms. Physiol Zool 69:403–417CrossRefGoogle Scholar
  11. Flik G, Kaneko T, Greco AM, Li J, Fenwick JC (1997) Sodium dependent ion transporters in trout gills. Fish Physiol Biochem 17:385–396CrossRefGoogle Scholar
  12. Franklin NM, Glover CN, Nicol JA, Wood CM (2005) Calcium/cadmium interactions at uptake surfaces in rainbow trout: Waterborne versus dietary routes of exposure. Environ Toxicol Chem 24:2954–2964CrossRefPubMedGoogle Scholar
  13. Grosell M (2010) The role of the gastrointestinal tract in salt and water balance. In: Grosell M, Farrell AP, Brauner CJ (eds) Fish physiology, vol 30. Academic Press, Amsterdam, pp 135–164Google Scholar
  14. Hanssen RGJM, Aarden EM, van der Venne WPHG, Pang PKT, Wendelaar Bonga SE (1991) Regulation of secretion of the teleost fish hormone stanniocalcin: effects of extracellular calcium. Gen Comp Endocrinol 163:155–163CrossRefGoogle Scholar
  15. Hogstrand C, Wilson RW, Polgar D, Wood CM (1994) Effects of zinc on the kinetics of branchial calcium uptake in freshwater rainbow trout during adaptation to waterborne zinc. J Exp Biol 186:55–73PubMedGoogle Scholar
  16. Hsu HH, Lin LY, Tseng YC, Horng JL, Hwang PP (2014) A new model for fish ion regulation: identification of ionocytes in freshwater- and seawater-acclimated medaka (Oryzias latipes). Cell Tissue Res 357:225–243CrossRefPubMedGoogle Scholar
  17. Hwang PP, Lee TH (2007) New insights into fish ion regulation and mitochondrion-rich cells. Comp Biochem Physiol A 148:479–497CrossRefGoogle Scholar
  18. Kester DR, Duedall IW, Connors DN, Pytkowicz RM (1967) Preparation of artificial seawater. Limnol Oceanogr 12:176–179CrossRefGoogle Scholar
  19. Kwong RWM, Kumai Y, Perry SF (2016) Neuroendocrine control of ionic balance in zebrafish. Gen Comp Endocrinol 234:40–46CrossRefPubMedGoogle Scholar
  20. Liao BK, Deng AN, Chen SC, Chou MY, Hwang PP (2007) Expression and water calcium dependence of calcium transporter isoforms in zebrafish gill mitochondrion-rich cells. BMC Genom 8:1–13CrossRefGoogle Scholar
  21. Lin C-H, Hwang P-P (2016) The control of calcium metabolism in zebrafish (Danio rerio). Int J Mol Sci 17:1783CrossRefPubMedCentralGoogle Scholar
  22. Lin CH, Kuan WC, Liao BK, Deng AN, Tseng DY, Hwang PP (2016) Environmental and cortisol-mediated control of Ca2+ uptake in tilapia (Oreochromis mossambicus). J Comp Physiol B 186:323–332CrossRefPubMedPubMedCentralGoogle Scholar
  23. Malvin RL, Schiff D, Eiger S (1980) Angiotensin and drinking rates in the euryhaline killifish. Am J Physiol Regul Integr Comp Physiol 239:R31–R34CrossRefGoogle Scholar
  24. Marshall WS (2002) Na+, Cl, Ca2+ and Zn2+ transport by fish gills: retrospective review and prospective synthesis. J Exp Zool 293:264–283CrossRefPubMedGoogle Scholar
  25. Marshall WS, Bryson SE, Burghardt JS, Verbost PM (1995) Ca2+ transport by opercular epithelium of the fresh water adapted euryhaline teleost, Fundulus heteroclitus. J Comp Physiol B 165:268–277CrossRefGoogle Scholar
  26. Marshall W, Emberley T, Singer T, Bryson S, Mccormick S (1999) Time course of salinity adaptation in a strongly euryhaline estuarine teleost, Fundulus heteroclitus: a multivariable approach. J Exp Biol 202:1535–1544PubMedGoogle Scholar
  27. Mayer-Gostan N, Bornancin M, DeRenzis G, Naon R, Yee JA, Shew RL, Pang PK (1983) Extraintestinal calcium uptake in the killifish, Fundulus heteroclitus. J Exp Zool 227:329–338CrossRefPubMedGoogle Scholar
  28. Niyogi S, Wood CM (2004) Kinetic analyses of waterborne Ca and Cd transport and their interactions in the gills of rainbow trout (Oncorhynchus mykiss) and yellow perch (Perca flavescens), two species differing greatly in acute waterborne Cd sensitivity. J Comp Physiol B 174:243–253CrossRefPubMedGoogle Scholar
  29. Pan T-C, Liao B-K, Huang C-J, Lin L-Y, Hwang P-P (2005) Epithelial Ca2+ channel expression and Ca2+ uptake in developing zebrafish. Am J Physiol Regul Integr Comp Physiol 289:R1202–R1211CrossRefPubMedGoogle Scholar
  30. Pang PKT, Pang RK (1986) Hormones and calcium regulation in Fundulus heteroclitus. Am Zool 234:225–234CrossRefGoogle Scholar
  31. Pang PKT, Griffith RW, Maetz J, Pic R (1980) Calcium uptake in fishes. In: Lahlou B (ed) Epithelial transport in lower vertebrates. Cambridge University Press, Cambridge, pp 122–132Google Scholar
  32. Patrick ML, Wood CM, Marshall WS (1997) Calcium regulation in the freshwater-adapted mummichog. J Fish Biol 51:135–145CrossRefPubMedGoogle Scholar
  33. Perry SF (1997) The chloride cell: structure and function in the gills of freshwater fishes. Annu Rev Physiol 59:325–347CrossRefPubMedGoogle Scholar
  34. Perry SF, Flik G (1988) Characterization of branchial transepithelial calcium fluxes in freshwater trout, Salmo gairdneri. Am J Physiol Regul Integr Comp Physiol 254:R491–R498CrossRefGoogle Scholar
  35. Perry SF, Wood CM (1985) Kinetics of branchial calcium uptake in the rainbow trout: effects of acclimation to various external calcium levels. J Exp Biol 116:411–433Google Scholar
  36. Perry SF, Shahsavarani A, Georgalis T, Bayaa M, Furimsky M, Thomas SLY (2003) Channels, pumps, and exchangers in the gill and kidney of freshwater fishes: their role in ionic and acid–base regulation. J Exp Zool 300A:53–62CrossRefGoogle Scholar
  37. Potts WTW, Evans DH (1967) Sodium and chloride balance in the killifish Fundulus heteroclitus. Biol Bull 133:411–425CrossRefPubMedGoogle Scholar
  38. Prodocimo V, Galvez F, Freire CA, Wood CM (2007) Unidirectional Na+ and Ca2+ fluxes in two euryhaline teleost fishes, Fundulus heteroclitus and Oncorhynchus mykiss, acutely submitted to a progressive salinity increase. J Comp Physiol B 177:519–528CrossRefPubMedGoogle Scholar
  39. Qiu A, Hogstrand C (2004) Functional characterisation and genomic analysis of an epithelial calcium channel (ECaC) from pufferfish, Fugu rubripes. Gene 342:113–123CrossRefPubMedGoogle Scholar
  40. Rogers JT, Wood CM (2004) Characterization of branchial lead-calcium interaction in the freshwater rainbow trout Oncorhynchus mykiss. J Exp Biol 207:813–825CrossRefPubMedGoogle Scholar
  41. Schoenmakers TJM, Verbost PM, Flik G, Wendelaar Bonga SE (1993) Transcellaular intestinal calcium transport in freshwater and seawater fish and its dependence on sodium/calcium exchange. J Exp Biol 176:195–206Google Scholar
  42. Scott GR, Richards JG, Forbush B, Isenring P, Schulte PM (2004) Changes in gene expression in gills of the euryhaline killifish Fundulus heteroclitus after abrupt salinity transfer. Am J Physiol Cell Physiol 287:C300–C309CrossRefPubMedGoogle Scholar
  43. Scott GR, Claiborne JB, Edwards SL, Schulte PM, Wood CM (2005) Gene expression after freshwater transfer in gills and opercular epithelia of killifish: insight into divergent mechanisms of ion transport. J Exp Biol 208:2719–2729CrossRefPubMedGoogle Scholar
  44. Scott GR, Schulte PM, Wood CM (2006) Plasticity of osmoregulatory function in the killifish intestine: drinking rates, salt and water transport, and gene expression after freshwater transfer. J Exp Biol 209:4040–4050CrossRefPubMedGoogle Scholar
  45. Scott GR, Baker DW, Schulte PM, Wood CM (2008) Physiological and molecular mechanisms of osmoregulatory plasticity in killifish after seawater transfer. J Exp Biol 211:2450–2459CrossRefPubMedGoogle Scholar
  46. Shahsavarani A, Perry SF (2006) Hormonal and environmental regulation of epithelial calcium channel in gill of rainbow trout (Oncorhynchus mykiss). Am J Physiol Regul Integr Comp Physiol 291:1490–1498CrossRefGoogle Scholar
  47. Shahsavarani A, McNeil B, Galvez F, Wood CM, Goss GG, Hwang P-P, Perry SF (2006) Characterization of a branchial epithelial calcium channel (ECaC) in freshwater rainbow trout (Oncorhynchus mykiss). J Exp Biol 209:1928–1943CrossRefPubMedGoogle Scholar
  48. Sundell K, Bjornsson BT (1988) Kinetics of calcium fluxes across the intestinal mucosa of the marine teleost, Gadus morhua, measured using an in vitro perfusion method. J Exp Biol 140:171–186Google Scholar
  49. Verbost PM, Schoenmakers TJ, Flik G, Wendelaar Bonga SE (1994) Kinetics of ATP- and Na+-gradient driven Ca2+ transport in basolateral membranes from gills of freshwater- and seawater-adapted tilapia. J Exp Biol 186:95–108PubMedGoogle Scholar
  50. Verbost PM, Bryson SE, Wendelaar Bonga SE, Marshall WS (1997) Na+-dependent Ca2+ uptake in isolated opercular epithelium of Fundulus heteroclitus. J Comp Physiol B 167:205–212CrossRefPubMedGoogle Scholar
  51. Wendelaar Bonga SE, Pang PKT (1991) Control of calcium regulating hormones in the vertebrates: parathyroid hormone, calcitonin, prolactin, and stanniocalcin. Int Rev Cytol 128:139–213CrossRefPubMedGoogle Scholar
  52. Wood CM (2011) Rapid regulation of Na+ and Cl flux rates in killifish after acute salinity challenge. J Exp Mar Biol Ecol 409:62–69CrossRefGoogle Scholar
  53. Wood CM, Grosell M (2008) A critical analysis of transepithelial potential in intact killifish (Fundulus heteroclitus) subjected to acute and chronic changes in salinity. J Comp Physiol B 178:713–727CrossRefPubMedGoogle Scholar
  54. Wood CM, Laurent P (2003) Na+ versus Cl transport in the intact killifish after rapid salinity transfer. Biochim Biophys Acta Biomembr 1618:106–119CrossRefGoogle Scholar
  55. Wood CM, Marshall WS (1994) Ion balance, acid-base regulation, and chloride cell function in the common killifish, Fundulus heteroclitus—a euryhaline estuarine teleost. Estuaries 17:34–52CrossRefGoogle Scholar

Copyright information

© Springer-Verlag GmbH Germany, part of Springer Nature 2018

Authors and Affiliations

  1. 1.Department of BiologyUniversity of OttawaOttawaCanada
  2. 2.EcoToxMiamiUSA
  3. 3.Rosenstiel School of Marine Atmospheric ScienceUniversity of MiamiMiamiUSA
  4. 4.Department of BiologyMcMaster UniversityHamiltonCanada
  5. 5.Department of ZoologyUniversity of British ColumbiaVancouverCanada

Personalised recommendations