Journal of Comparative Physiology B

, Volume 186, Issue 5, pp 589–602 | Cite as

The response of claudin-like transmembrane septate junction proteins to altered environmental ion levels in the larval mosquito Aedes aegypti

  • Sima Jonusaite
  • Scott P. Kelly
  • Andrew Donini
Original Paper


Septate junctions (SJs) occlude the paracellular pathway and function as paracellular diffusion barriers within invertebrate epithelia. However, integral components of SJs and their contribution to barrier properties have received considerably less attention than those of vertebrate occluding junctions. In arthropods, SJ proteins have only been identified in Drosophila and among these are three integral claudin-like proteins, Megatrachea (Mega), Sinuous (Sinu) and Kune-kune (Kune), as well as a receptor-like transmembrane SJ protein known as Neurexin IV (Nrx IV). In this study, mega, sinu, kune and nrx IV are identified and characterized in aquatic larvae of the mosquito Aedes aegypti and a role for these proteins in ionoregulatory homeostasis is considered. Transcripts encoding Mega, Sinu, Kune and Nrx IV were found in iono/osmoregulatory tissues such as the midgut, Malpighian tubules, hindgut and anal papillae, but abundance was greater in the hindgut and anal papillae. Using immunohistochemical and western blot analysis it was found that Kune localized to the regions of intercellular contact between epithelial cells of the rectum and posterior midgut and in the apical membrane domain of the syncytial epithelium of anal papillae. To investigate a potential role for integral SJ proteins in larval A. aegypti iono/osmoregulation, abundance was examined in animals reared in freshwater or brackish water (30 % seawater). In iono/osmoregulatory epithelia, larvae exhibited tissue-specific alterations in mega mRNA and Kune protein abundance, but not sinu or nrx IV mRNA. These studies provide a first look at the potential contribution of integral SJ components to iono/osmoregulatory homeostasis in an aquatic invertebrate.


Mosquito Osmoregulation Septate junctions Claudins Neurexin IV Salinity 



This study was supported by a Natural Sciences and Engineering Research Council of Canada Discovery Grants to SPK and AD, an Ontario Graduate Scholarship to SJ. The authors would like to thank Dr. Mikio Furuse for anti-Kune antibody.

Compliance with ethical standards

Competing interests

No competing interests declared.


  1. Anderson JM, Van Itallie M (2009) Physiology and function of the tight junction. Cold Spring Harb Perspect Biol 1:a002584CrossRefPubMedPubMedCentralGoogle Scholar
  2. Angelow S, Ahlstrom R, Yu ASL (2008) Biology of claudins. Am J Physiol Renal Physiol 295:F867–F876CrossRefPubMedPubMedCentralGoogle Scholar
  3. Asano A, Asano K, Sasaki H, Furuse M, Tsukita S (2003) Claudins in Caenorhabditis elegans: their distribution and barrier function in the epithelium. Curr Biol 13:1042–1046CrossRefPubMedGoogle Scholar
  4. Bagherie-Lachidan M, Wright SI, Kelly SP (2008) Claudin-3 tight junction proteins in Tetraodon nigroviridis: cloning, tissue-specific expression, and a role in hydromineral balance. Am J Physiol Regul Integr Comp Physiol 294:R1638–R1647CrossRefPubMedGoogle Scholar
  5. Baumann O (2001) Posterior midgut epithelial cells differ in their organization of the membrane skeleton from other Drosophila epithelia. Exp Cell Res 270:176–187CrossRefPubMedGoogle Scholar
  6. Baumgartner S, Littleton JT, Broadie K, Bhat MA, Harbecke R, Lengyel JA, Chiquet-Ehrismann R, Prokop A, Bellen HJ (1996) A Drosophila neurexin is required for septate junction and blood-nerve barrier formation and function. Cell 87:1059–1068CrossRefPubMedGoogle Scholar
  7. Behr M, Riedel D, Schuh R (2003) The claudin-like Megatrachea is essential in septate junctions for the epithelial barrier function in Drosophila. Dev Cell 5:611–620CrossRefPubMedGoogle Scholar
  8. Beyenbach KW, Piermarini PM (2011) Transcellular and paracellular pathways of transepithelial fluid secretion in Malpighian (renal) tubules of the yellow fever mosquito Aedes aegypti. Acta Physiol (Oxf) 202:387–407. doi: 10.1111/j.1748-1716.2010.02195.x CrossRefGoogle Scholar
  9. Bradley TJ (1994) The role of physiological capacity, morphology, and phylogeny in determining habitat use in mosquitoes. In: Wainwright PC, Reilly SM (eds) Ecological morphology. The University of Chicago Press, Chicago, pp 303–318Google Scholar
  10. Bui P, Kelly SP (2014) Claudin-6, -10d, and -10e contribute to seawater acclimation in the euryhaline puffer fish Tetraodon nigroviridis. J Exp Biol 217:1758–1767CrossRefPubMedGoogle Scholar
  11. Byri S, Misra T, Syed ZA, Bätz T, Shah J, Boril L, Glashauser J, Aegerter-Wilmsen T, Matzat T, Moussian B, Uv A, Luschnig S (2015) The triple-repeat protein anakonda controls epithelial tricellular junction formation in Drosophila. Dev Cell 33:535–548. doi: 10.1016/j.devcel.2015.03.023 CrossRefPubMedGoogle Scholar
  12. Chasiotis H, Kelly SP (2008) Occludin immunolocalization and protein expression in goldfish. J Exp Biol 211:1524–1534CrossRefPubMedGoogle Scholar
  13. Chasiotis H, Kelly SP (2009) Occludin and hydromineral balance in Xenopus laevis. J Exp Biol 212:287–296CrossRefPubMedGoogle Scholar
  14. Chasiotis H, Kolosov D, Kelly SP (2012a) Permeability properties of the teleost fish gill epithelium under ion-poor conditions. Am J Physiol Regul Integr Comp Physiol 302:R727–R739CrossRefPubMedGoogle Scholar
  15. Chasiotis H, Kolosov D, Bui P, Kelly SP (2012b) Tight junctions, tight junction proteins and paracellular permeability across the gill epithelium of fishes: a review. Resp Physiol Neurobiol 184:269–281CrossRefGoogle Scholar
  16. Clark TM, Koch A, Moffett DF (1999) The anterior and posterior ʻstomach’ regions of larval Aedes aegypti midgut: regional specialization of ion transport and stimulation by 5-hydroxytryptamine. J Exp Biol 202:247–252PubMedGoogle Scholar
  17. Clark TM, Koch A, Moffett DF (2000) The electrical properties of the anterior stomach of the larval mosquito (Aedes aegypti). J Exp Biol 203:1093–1101PubMedGoogle Scholar
  18. Clark TM, Hutchinson MJ, Huegel KL, Moffett SB, Moffett DF (2005) Additional morphological and physiological heterogeneity within the midgut of larval Aedes aegypti (Diptera: Culicidae) revealed by histology, electrophysiology, and effects of Bacillus thuringiensis endotoxin. Tissue Cell 37:457–468CrossRefPubMedGoogle Scholar
  19. Clements AN (1992) The biology of mosquitoes, vol 1. Chapman & Hall, LondonGoogle Scholar
  20. Del Duca O, Nasirian A, Galperin V, Donini A (2011) Pharmacological characterisation of apical Na+ and Cl transport mechanisms of the anal papillae in the larval mosquito Aedes aegypti. J Exp Biol 214:3992–3999. doi: 10.1242/jeb.063719 CrossRefPubMedGoogle Scholar
  21. Donini A, O’Donnell MJ (2005) Analysis of Na+, Cl, K+, H+ and NH4 + concentration gradients adjacent to the surface of anal papillae of the mosquito Aedes aegypti: application of self-referencing ion-selective microelectrodes. J Exp Biol 208:603–610CrossRefPubMedGoogle Scholar
  22. Donini A, Patrick ML, Bijelic G, Christensen RJ, Ianowski JP, Rheault MR, O’Donnell MJ (2006) Secretion of water and ions by Malpighian tubules of larval mosquitoes: effects of diuretic factors, second messengers, and salinity. Physiol Biochem Zool 79:645–655CrossRefPubMedGoogle Scholar
  23. Donini A, Gaidhu MP, Strasberg D, O’Donnell MJ (2007) Changing salinity induces alterations in hemolymph ion concentrations and Na+ and Cl transport kinetics of the anal papillae in the larval mosquito, Aedes aegypti. J Exp Biol 210:983–992CrossRefPubMedGoogle Scholar
  24. Duffy NM, Bui P, Bagherie-Lachidan M, Kelly SP (2011) Epithelial remodeling and claudin mRNA abundance in the gill and kidney of puffer fish (Tetraodon biocellatus) acclimated to altered environmental ion levels. J Comp Physiol B 181:219–238CrossRefPubMedGoogle Scholar
  25. Edwards HA (1982) Aedes aegypti: energetics of osmoregulation. J Exp Biol 101:135–141Google Scholar
  26. Edwards HA, Harrison JB (1983) An osmoregulatory syncytium and associated cells in a freshwater mosquito. Tissue Cell 15:271–280CrossRefPubMedGoogle Scholar
  27. Flower NE, Filshie BK (1975) Junctional structures in the midgut cells of lepidopteran caterpillars. J Cell Sci 17:221–239PubMedGoogle Scholar
  28. Fournier ML, Paulson A, Pavelka N, Mosley AL, Gaudenz K, Bradford WD, Glynn E, Li H, Sardiu ME, Fleharty B, Seidel C, Florens L, Washburn MP (2010) Delayed correlation of mRNA and protein expression in rapamycin-treated cells and a role for Ggc1 in cellular sensitivity to rapamycin. Mol Cell Proteomics 9:271–284. doi: 10.1074/mcp.M900415-MCP200 CrossRefPubMedGoogle Scholar
  29. Furuse M, Sasaki H, Tsukita S (1999) Manner of interaction of heterogeneous claudin species within and between tight junction strands. J Cell Biol 147:891–903CrossRefPubMedPubMedCentralGoogle Scholar
  30. Furuse M, Hata M, Furuse K, Yoshida Y, Haratake A, Sugitani Y, Noda T, Kubo A, Tsukita S (2002) Claudin-based tight junctions are crucial for the mammalian epidermal barrier: a lesson from claudin-1-deficient mice. J Cell Biol 156:1099–1111CrossRefPubMedPubMedCentralGoogle Scholar
  31. Ganot P, Zoccola D, Tambutté E, Voolstra CR, Aranda M, Allemand D, Tambutté S (2015) Structural molecular components of septate junctions in cnidarians point to the origin of epithelial junctions in eukaryotes. Mol Biol Evol 32:44–62. doi: 10.1093/molbev/msu265 CrossRefPubMedGoogle Scholar
  32. Green CR, Bergquist PR (1982) Phylogentic relationships within the invertebrates in relation to the structure of septate junctions and the development of ‘occluding’ junctional types. J Cell Sci 53:279–305Google Scholar
  33. Gregory M, Dufresne J, Hermo L, Cyr D (2001) Claudin-1 is not restricted to tight junctions in the rat epididymis. Endocrinology 142:854–863PubMedGoogle Scholar
  34. Günzel D, Yu ASL (2013) Claudins and the modulation of tight junction permeability. Physiol Rev 93:525–569. doi: 10.1152/physrev.00019.2012 CrossRefPubMedPubMedCentralGoogle Scholar
  35. Ionescu A, Donini A (2012) Aedes CAPA-PVK-1 displays diuretic and dose dependent antidiuretic potential in the larval mosquito Aedes aegypti (Liverpool). J Insect Physiol 58:1299–1306CrossRefPubMedGoogle Scholar
  36. Izumi Y, Furuse M (2014) Molecular organization and function of invertebrate occluding junctions. Semin Cell Dev Biol 36:186–193CrossRefPubMedGoogle Scholar
  37. Izumi Y, Yanagihashi Y, Furuse M (2012) A novel protein complex, Mesh-Ssk, is required for septate junction formation in the Drosophila midgut. J Cell Sci 125:4923–4933CrossRefPubMedGoogle Scholar
  38. Jagadeshwaran U, Onken H, Hardy M, Moffett SB, Moffett DF (2010) Cellular mechanisms of acid secretion in the posterior midgut of the larval mosquito (Aedes aegypti). J Exp Biol 213:295–300CrossRefPubMedGoogle Scholar
  39. Jaspers MH, Nolde K, Behr M, Joo SH, Plessmann U, Nikolov M, Urlaub H, Schuh R (2012) The claudin Megatrachea protein complex. J Biol Chem 287:36756–36765CrossRefPubMedPubMedCentralGoogle Scholar
  40. Jonusaite S, Kelly SP, Donini A (2011) The physiological response of larval Chironomus riparius (Meigen) to abrupt brackish water exposure. J Comp Physiol B 181:343–352CrossRefPubMedGoogle Scholar
  41. Jonusaite S, Kelly SP, Donini A (2013) Tissue-specific ionomotive enzyme activity and K+ reabsorption reveal the rectum as an important ionoregulatory organ in larval Chironomus riparius exposed to varying salinity. J Exp Biol 216:3637–3648. doi: 10.1242/jeb.089219 CrossRefPubMedGoogle Scholar
  42. Jonusaite S, Donini A, Kelly SP (2016) Occluding junctions of invertebrate epithelia. J Comp Physiol B 186:17–43CrossRefPubMedGoogle Scholar
  43. Kaushal SS, Groffman PM, Likens GE, Belt KT, Stack WP, Kelly VR, Band LE, Fisher GT (2005) Increased salinization of fresh water in the northeastern United States. PNAS 102:13517–13520CrossRefPubMedPubMedCentralGoogle Scholar
  44. Knust E, Bossinger O (2002) Composition and formation of intercellular junctions in epithelial cells. Science 298:1955–1959CrossRefPubMedGoogle Scholar
  45. Kolosov D, Kelly SP (2013) A role for tricellulin in the regulation of gill epithelium permeability. Am J Physiol Regul Integr Comp Physiol 304:R1139–R1148CrossRefPubMedPubMedCentralGoogle Scholar
  46. Kolosov D, Bui P, Chasiotis H, Kelly SP (2013) Claudins in teleost fishes. Tissue Barriers 1:e25391. doi: 10.4161/tisb.25391 CrossRefPubMedPubMedCentralGoogle Scholar
  47. Kolosov D, Chasiotis H, Kelly SP (2014) Tight junction protein gene expression patterns and changes in transcript abundance during development of model fish gill epithelia. J Exp Biol 217:1667–1681CrossRefPubMedGoogle Scholar
  48. Kumai Y, Bahubeshi A, Steele S, Perry SF (2011) Strategies for maintaining Na+ balance in zebrafish (Danio rerio) during prolonged exposure to acid water. Comp Biochem Physiol 160A:52–62CrossRefGoogle Scholar
  49. Kwong RM, Perry SF (2013) The tight junction protein claudin-b regulates epithelial permeability and sodium handling in larval zebrafish, Danio rerio. Am J Physiol Regul Integr Comp Physiol 304:R504–R513CrossRefPubMedPubMedCentralGoogle Scholar
  50. Lane NJ, Skaer HB (1980) Intercellular junctions in insect tissues. In: Berridge MJ, Treherne JE, Wigglesworth VB (eds) Advances in insect physiology, vol 15. Academic Press, London, pp 35–213Google Scholar
  51. Laprise P, Lau KM, Harris KP, Silva-Gagliardi NF, Paul SM, Beronja S, Beitel GJ, McGlade CJ, Tepass U (2009) Yurt, Coracle, Neurexin IV and the Na(+), K(+)-ATPase form a novel group of epithelial polarity proteins. Nature 459:1141–1145. doi: 10.1038/nature08067 CrossRefPubMedGoogle Scholar
  52. Laprise P, Paul SM, Boulanger J, Robbins RM, Beitel GJ, Tepass U (2010) Epithelial polarity proteins regulate Drosophila tracheal tube size in parallel to the luminal matrix pathway. Curr Biol 20:55–61CrossRefPubMedPubMedCentralGoogle Scholar
  53. Littleton JT, Bhat MA, Bellen HJ (1997) Deciphering the function of neurexins at cellular junctions. J Cell Biol 137:793–796CrossRefPubMedPubMedCentralGoogle Scholar
  54. Luquet C, Pellerano G, Rosa G (1997) Salinity-induced changes in the fine structure of the gills of the semiterrestrial estuarian crab, Uca uruguayensis (Nobili, 1901) (Decapoda, Ocypodidae). Tissue Cell 29:495–501CrossRefPubMedGoogle Scholar
  55. Luquet CM, Genovese G, Rosa GA, Pellerano GN (2002) Ultrastructural changes in the gill epithelium of the crab Chasmagnathus granulates (Decapoda: Grapsidae) in diluted and concentrated seawater. Mar Bio 141:753–760CrossRefGoogle Scholar
  56. Nelson KS, Furuse M, Beitel GJ (2010) The Drosophila Claudin Kune-kune is required for septate junction organization and tracheal tube size control. Genetics 185:831–839CrossRefPubMedPubMedCentralGoogle Scholar
  57. Noirot-Timothee C, Noirot C (1980) Septate and scalariform junctions in arthropods. Int Rev Cytol 63:97–141CrossRefPubMedGoogle Scholar
  58. Onken H, Moffett DF (2009) Revisiting the cellular mechanisms of strong luminal alkalinization in the anterior midgut of larval mosquitoes. J Exp Biol 212:373–377CrossRefPubMedPubMedCentralGoogle Scholar
  59. Patrick ML, Bradley TJ (2000) The physiology of salinity tolerance in larvae of two species of Culex mosquitoes: the role of compatible solutes. J Exp Biol 203:821–830PubMedGoogle Scholar
  60. Patrick ML, Gonzalez RJ, Bradley TJ (2001) Sodium and chloride regulation in freshwater and osmoconforming larvae of Culex mosquitoes. J Exp Biol 204:3345–3354PubMedGoogle Scholar
  61. Patrick ML, Aimanova K, Sanders HR, Gill SS (2006) P-type Na+/K+-ATPase and V-type H+-ATPase expression patterns in the osmoregulatory organs of larval and adult mosquito Aedes aegypti. J Exp Biol 209:4638–4651CrossRefPubMedGoogle Scholar
  62. Pond GJ, Passmore ME, Borsuk FA, Reynolds L, Rose CA (2008) Downstream effects of moutaintop coal mining: comparing biological conditions using family and genus-level macroinvertebrate bioassessment tools. J North Am Benthol Soc 127:717–737CrossRefGoogle Scholar
  63. Ramasamy R, Surendran SN (2012) Global climate change and its potential impact on disease transmission by salinity-tolerant mosquito vectors in coastal zones. Front Physiol 3:198. doi: 10.3389/fphys.2012.00198 CrossRefPubMedPubMedCentralGoogle Scholar
  64. Simske JS, Hardin J (2011) Claudin family proteins in Caenorhabditis elegans. Methods Mol Biol 762:147–169. doi: 10.1007/978-1-61779-185-7_11 CrossRefPubMedGoogle Scholar
  65. Sohal RS, Copeland E (1966) Ultrastructural variations in the anal papillae of Aedes aegypti (L) at different environmental salinities. J Insect Physiol 12:429–434CrossRefPubMedGoogle Scholar
  66. Stork T, Engelen D, Krudewig A, Silies M, Bainton RJ, Klämbt C (2008) Organization and function of the blood-brain barrier in Drosophila. J Neurosci 28:587–597CrossRefPubMedGoogle Scholar
  67. Suzuki H, Ito Y, Yamazaki Y, Mineta K, Uji M, Abe K, Tani K, Fujiyoshi Y, Tsukita S (2013) The four-transmembrane protein IP39 of Euglena forms strands by a trimeric unit repeat. Nat Commun 4:1766. doi: 10.1038/ncomms2731 CrossRefPubMedPubMedCentralGoogle Scholar
  68. Tepass U, Tanentzapf G, Ward R, Fehon R (2001) Epithelial cell polarity and cell junctions in Drosophila. Annu Rev Genet 35:747–784CrossRefPubMedGoogle Scholar
  69. Tipsmark CK, Kiilerich P, Nilsen TO, Ebbesson LOE, Stefansson SO, Madsen SS (2008) Branchial expression patterns of claudin isoforms in Atlantic salmon during seawater acclimation and smoltification. Am J Physiol Integr Regul Comp Physiol 294:R1563–R1574CrossRefGoogle Scholar
  70. Van Itallie CM, Anderson JM (2013) Claudin interactions in and out of the tight junction. Tissue Barriers 1:e25247. doi: 10.4161/tisb.25247 CrossRefPubMedPubMedCentralGoogle Scholar
  71. Williams WD (2001) Anthropogenic salinization of inland waters. Hydrobiol 466:329–337CrossRefGoogle Scholar
  72. Wu VM, Schulte J, Hirschi A, Tepass U, Beitel GJ (2004) Sinuous is a Drosophila claudin required for septate junction organization and epithelial tube size control. J Cell Biol 164:313–323CrossRefPubMedPubMedCentralGoogle Scholar
  73. Yanagihashi Y, Usui T, Izumi Y, Yonemura S, Sumida M, Tsukita S, Uemura T, Furuse M (2012) Snakeskin, a membrane protein associated with smooth septate junctions, is required for intestinal barrier function in Drosophila. J Cell Sci 125:1980–1990CrossRefPubMedGoogle Scholar

Copyright information

© Springer-Verlag Berlin Heidelberg 2016

Authors and Affiliations

  • Sima Jonusaite
    • 1
  • Scott P. Kelly
    • 1
  • Andrew Donini
    • 1
  1. 1.Department of Biology, 205 LumbersYork UniversityTorontoCanada

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