SPANINGS-PIERROT, C.; WEIHRAUCH, D.*; LIGNOT, J.-H.; CHARMANTIER, G.; TOWLE, D.W.: Immunolocalization and gene expression of Na,K-ATPase in gills of the grapsid crab Pachygrapsus marmoratus .

The activity, immunolocalization, and mRNA expression of Na,K-ATPase were studied in gills of the hyper-/hypoosmoregulating crab Pachygrapsus marmoratus acclimated to salinities of 10, 37, and 45 ppt. Enzyme activity was significantly higher in pooled posterior gills (7-9) than in anterior gills (1-6) and increased significantly upon acclimation to diluted medium, confirming previous observations with other crab species. Immunofluorescent analysis of gill sections indicated localization of Na,K-ATPase protein in basolateral membranes of branchial epithelium adjacent to the afferent blood vessel. Using degenerate primers based on Na,K-ATPase ALPHA subunit homologies published in GenBank, we amplified related sequences in cDNA transcribed from gill mRNA and employed the resulting PCR products for sequencing and mRNA expression analyses. Immunofluorescence and mRNA quantitation produced parallel results, showing high expression of Na,K-ATPase protein and mRNA in gills 6, 7, and 8 at low salinity. In gills 6 and 8, the abundance of Na,K-ATPase protein and mRNA increased dramatically with decreasing salinity. However, gill 7 showed substantial levels of Na,K-ATPase protein and mRNA even in 45 ppt seawater, suggesting that this gill may play an important role in hypoosmoregulation. Our results offer a molecular explanation for the previously observed changes in Na,K-ATPase activity in gills of euryhaline crabs adjusting to osmoregulatory demands in salinity extremes, namely the upregulation of Na,K-ATPase gene transcription and subsequent translation of Na,K-ATPase protein. Supported by NSF (IBN-9807539).