Abstract
Mummichogs (Fundulus heteroclitus) can tolerate abrupt changes in environmental salinity because of their ability to rapidly adjust the activities of ionocytes in branchial and opercular epithelia. In turn, the concerted expression of sub-cellular effectors of ion transport underlies adaptive responses to fluctuating salinities. Exposure to seawater (SW) stimulates the expression of Na+/K+/2Cl− cotransporter 1 (nkcc1) and cystic fibrosis transmembrane regulator (cftr) mRNAs in support of ion extrusion by SW-type ionocytes. Given the incomplete understanding of how freshwater (FW)-type ionocytes actually operate in mummichogs, the transcriptional responses essential for ion absorption in FW environments remain unresolved. In a subset of species, a ‘fish-specific’ Na+/Cl− cotransporter denoted Ncc2 (Slc12a10) is responsible for the uptake of Na+ and Cl− across the apical surface of FW-type ionocytes. In the current study, we identified an ncc2 transcript that is highly expressed in gill filaments and opercular epithelium of FW-acclimated mummichogs. Within 1 day of transfer from SW to FW, ncc2 levels in both tissues increased in parallel with reductions in nkcc1 and cftr. Conversely, mummichogs transferred from FW to SW exhibited marked reductions in ncc2 concurrent with increases in nkcc1 and cftr. Immunohistochemical analyses employing a homologous antibody revealed apical Ncc2-immunoreactivity in Na+/K+-ATPase-immunoreactive ionocytes of FW-acclimated animals. Our combined observations suggest that Ncc2/ncc2-expressing ionocytes support the capacity of mummichogs to inhabit FW environments.
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References
Able KW (2002) Killifishes. In: Collette BB, Klein-MacPhee G (eds) Bigelow and Schroeder’s fishes of the Gulf of Maine, 3rd edn. Smithsonian Institution Press, Washington, pp 292–297
Altschul SF, Madden TL, Schäffer AA, Zhang J, Zhang Z, Miller W, Lipman DJ (1997) Gapped BLAST and PSI-BLAST: a new generation of protein database search programs. Nucleic Acids Res 25:3389–3402
Benson DA, Karsch-Mizrachi I, Lipman DJ, Ostell J, Rapp BA, Wheeler DL (2000) GenBank. Nucleic Acids Res 28:15–18
Bollinger RJ, Madsen SS, Bossus MC, Tipsmark CK (2016) Does Japanese medaka (Oryzias latipes) exhibit a gill Na+/K+-ATPase isoform switch during salinity change? J Comp Physiol B 186:485–501
Brennan RS, Galvez F, Whitehead A (2015) Reciprocal osmotic challenges reveal mechanisms of divergence in phenotypic plasticity in the killifish Fundulus heteroclitus. J Exp Biol 218:1212–1222
Breves JP, Watanabe S, Kaneko T, Hirano T, Grau EG (2010) Prolactin restores branchial mitochondrion-rich cells expressing Na+/Cl− cotransporter in hypophysectomized Mozambique tilapia. Am J Physiol Regul Integr Comp Physiol 299:R702–710
Burden CE (1956) The failure of hypophysectomized Fundulus heteroclitus to survive in fresh water. Biol Bull 110:8–28
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 2:257–286
Chang WJ, Wang YF, Hu HJ, Wang JH, Lee TH, Hwang PP (2013) Compensatory regulation of Na+ absorption by Na+/H+ exchanger and Na+–Cl− cotransporter in zebrafish (Danio rerio). Front Zool 10:46
Dymowska AK, Hwang PP, Goss GG (2012) Structure and function of ionocytes in the freshwater fish gill. Respir Physiol Neurobiol 18:282–292
Felsenstein J (1989) PHYLIP–Phylogeny Inference Package (version 3.2). Cladistics 5:164–166
Fiol DF, Kültz D (2007) Osmotic stress sensing and signaling in fishes. FEBS J 274:5790–5798
Flemmer AW, Monette MY, Djurisic M, Dowd B, Darman R, Gimenez I, Forbush B (2010) Phosphorylation state of the Na+–K+–Cl− cotransporter (NKCC1) in the gills of Atlantic killifish (Fundulus heteroclitus) during acclimation to water of varying salinity. J Exp Biol 213:1558–1566
Griffith RW (1974) Environment and salinity tolerance in the genus Fundulus. Copeia 1974:319–331
Hiroi J, McCormick SD (2012) New insights into gill ionocyte and ion transporter function in euryhaline and diadromous fish. Respir Physiol Neurobiol 184:257–268
Hiroi J, Yasumasu S, McCormick SD, Hwang PP, Kaneko T (2008) Evidence for an apical Na–Cl cotransporter involved in ion uptake in a teleost fish. J Exp Biol 211:2584–2599
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 35:225–243
Inokuchi M, Hiroi J, Watanabe S, Lee KM, Kaneko T (2008) Gene expression and morphological localization of NHE3, NCC and NKCC1a in branchial mitochondria-rich cells of Mozambique tilapia (Oreochromis mossambicus) acclimated to a wide range of salinities. Comp Biochem Physiol A 151:151–158
Inokuchi M, Breves JP, Moriyama S, Watanabe S, Kaneko T, Lerner DT, Grau EG, Seale AP (2015) Prolactin 177, prolactin 188 and extracellular osmolality independently regulate the gene expression of ion transport effectors in gill of Mozambique tilapia. Am J Physiol Regul Integr Comp Physiol 309:R1251–1263
Karnaky KJ (1986) Structure and function of the chloride cell of Fundulus heteroclitus and other teleosts. Am Zool 26:209–224
Kato A, Muro T, Kimura Y, Li S, Islam Z, Ogoshi M, Doi H, Hirose S (2011) Differential expression of Na+-Cl− cotransporter and Na+-K+-Cl− cotransporter 2 in the distal nephrons of euryhaline and seawater pufferfishes. Am J Physiol Regul Integr Comp Physiol 300:R284–R297
Katoh F, Cozzi RR, Marshall WS, Goss GG (2008) Distinct Na+/K+/2Cl− cotransporter localization in kidneys and gills of two euryhaline species, rainbow trout and killifish. Cell Tissue Res 334:265–281
Kültz D (2012) The combinatorial nature of osmosensing in fishes. Physiology 27:259–275
Lytle C, Xu JC, Biemesderfer D, Forbush B (1995) Distribution and diversity of Na–K–Cl cotransport proteins: a study with monoclonal antibodies. Am J Physiol 269:C1496–1505
Marshall WS, Grosell M (2006) Ion transport, osmoregulation and acid–base balance. In: Evans DH, Claiborne JB (eds) The physiology of fishes. CRC Press, Boca Raton, pp 177–230
Marshall WS, Bryson SE, Darling P, Whitten C, Patrick M, Wilkie M, Wood CM, Buckland Nicks J (1997) NaCl transport and ultrastructure of opercular epithelium from a freshwater-adapted euryhaline teleost, Fundulus heteroclitus. J Exp Zool 277:23–37
Marshall WS, Bryson SE, Luby T (2000) Control of epithelial Cl− secretion by basolateral osmolality in the euryhaline teleost Fundulus heteroclitus. J Exp Biol 203:1897–1905
Marshall WS, Ossum CG, Hoffmann EK (2005) Hypotonic shock mediation by p38 MAPK, JNK, PKC, FAK, OSR1 and SPAK in osmosensing chloride secreting cells of killifish opercular epithelium. J Exp Biol 208:1063–1077
Marshall WS, Katoh F, Main HP, Sers N, Cozzi RR (2008) Focal adhesion kinase and β1 integrin regulation of Na+, K+, 2Cl− cotransporter in osmosensing ion transporting cells of killifish, Fundulus heteroclitus. Comp Biochem Physiol A 150:288–300
Marshall WS, Cozzi RRF, Spieker M (2017) WNK1 and p38-MAPK distribution in ionocytes and accessory cells of euryhaline teleost fish implies ionoregulatory function. Biol Open 6:956–966
Patrick ML, Wood CM (1999) Ion and acid–base regulation in the freshwater mummichog (Fundulus heteroclitus): a departure from the standard model for freshwater teleosts. Comp Biochem Physiol A 122:445–456
Patrick ML, Pärt P, Marshall WS, Wood CM (1997) Characterization of ion and acid–base transport in the freshwater adapted mummichog (Fundulus heteroclitus). J Exp Zool 279:208–219
Pérez-Ruis C, Gaitán-Peñas H, Estévez R, Barrallo-Gimeno A (2015) Identification and characterization of the zebrafish ClC-2 chloride channel orthologs. Pflügers Arch 467:1769–1781
Pfaffl MW (2001) A new mathematical model for relative quantification in real-time RT-PCR. Nucl Acids Res 29(9):e45
Pickford GE, Phillips JG (1959) Prolactin, a factor in promoting survival of hypophysectomized killifish in fresh water. Science 130:454–455
Schultz ET, McCormick SD (2013) Euryhalinity in an evolutionary context. In: McCormick SD, Farrell AP, Brauner CJ (eds) Euryhaline fishes. Elsevier, New York, pp 477–529
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–C309
Scott GR, Claiborne JB, Edwards SL, Schulte PM, Wood CB (2005) Gene expression after freshwater transfer in gills and opercular epithelia of killifish: insight into divergent mechanisms of ion transport. J Exp Biol 208:2719–2729
Takei Y, Hiroi J, Takahashi H, Sakamoto T (2014) Diverse mechanisms for body fluid regulation in teleost fishes. Am J Physiol Regul Integr Comp Physiol 307:R778–792
Teranishi K, Mekuchi M, Kaneko T (2013) Expression of sodium/hydrogen exchanger 3 and cation-chloride cotransporters in the kidney of Japanese eel acclimated to a wide range of salinities. Comp Biochem Physiol A 164:333–343
Tipsmark CK, Mahmmoud YA, Borski RJ, Madsen SS (2010) FXYD-11 associates with Na+-K+-ATPase in the gill of Atlantic salmon: regulation and localization in relation to changed ion-regulatory status. Am J Physiol Regul Integr Comp Physiol 299:R1212–1223
Wang YF, Tseng YC, Yan JJ, Hiroi J, Hwang PP (2009) Role of SLC12A10.2, a Na–Cl cotransporter-like protein, in a Cl uptake mechanism in zebrafish (Danio rerio). Am J Physiol Regul Integr Comp Physiol 296:R1650–1660
Wang YF, Yan JJ, Tseng YC, Chen RD, Hwang PP (2015) Molecular physiology of an extra-renal Cl− uptake mechanism for body fluid Cl− homeostasis. Int J Biol Sci 11:1190–1203
Whitehead A (2010) The evolutionary radiation of diverse osmotolerant physiologies in killifish (Fundulus sp.). Evolution 64:2070–2085
Wood CM, Laurent P (2003) Na+ versus Cl– transport in the intact killifish after rapid salinity transfer. Biochim Biophys Acta 1618:106–119
Yan JJ, Hwang PP (2019) Novel discoveries in acid–base regulation and osmoregulation: a review of selected hormonal actions in zebrafish and medaka. Gen Comp Endocrinol 277:20–29
Zadunaisky JA, Cardona S, Au L, Roberts DM, Fisher E, Lowenstein B, Cragoe EJ, Spring KR (1995) Chloride transport activation by plasma osmolarity during rapid adaptation to high salinity of Fundulus heteroclitus. J Membr Biol 143:207–217
Acknowledgements
We are grateful to Kirsten Tomlinson for collecting animals included in this report. We appreciate the excellent fish care provided by Kristin Salisbury, Aaron Cordiale, and Tracy Broderson. Jennifer Bonner and Eleanore Ritter provided valuable assistance with immunohistochemistry and confocal imaging.
Funding
This study was supported by the National Science Foundation (IOS-1755131 to J.P.B and IOS-1251616 to C.K.T.), Arkansas Biosciences Institute (C.K.T) and Skidmore College (Start-Up Funds to J.P.B.).
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Breves, J.P., Starling, J.A., Popovski, C.M. et al. Salinity-dependent expression of ncc2 in opercular epithelium and gill of mummichog (Fundulus heteroclitus). J Comp Physiol B 190, 219–230 (2020). https://doi.org/10.1007/s00360-020-01260-x
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DOI: https://doi.org/10.1007/s00360-020-01260-x