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Bossus, M., Charmantier, G., Blondeau-Bidet, E., Valletta, B., Boulo, V., & Lorin-Nebel, C. (2013). The ClC-3 chloride channel and osmoregulation in the European Sea Bass, Dicentrarchus labrax. J Comp Physiol B, 183(5), 641–662.
Résumé: Dicentrarchus labrax migrates between sea (SW), brackish and fresh water (FW) where chloride concentrations and requirements for chloride handling change: in FW, fish absorb chloride and restrict renal losses; in SW, they excrete chloride. In this study, the expression and localization of ClC-3 and Na(+)/K(+)-ATPase (NKA) were studied in fish adapted to SW, or exposed to FW from 10 min to 30 days. In gills, NKA-alpha1 subunit expression transiently increased from 10 min and reached a stabilized intermediate expression level after 24 h in FW. ClC-3 co-localized with NKA in the basolateral membrane of mitochondria-rich cells (MRCs) at all conditions. The intensity of MRC ClC-3 immunostaining was significantly higher (by 50 %) 1 h after the transfer to FW, whereas the branchial ClC-3 protein expression was 30 % higher 7 days after the transfer as compared to SW. This is consistent with the increased number of immunopositive MRCs (immunostained for NKA and ClC-3). However, the ClC-3 mRNA expression was significantly lower in FW gills. In the kidney, after FW transfer, a transient decrease in NKA-alpha1 subunit expression was followed by significantly higher stable levels from 24 h. The low ClC-3 protein expression detected at both salinities was not observed by immunocytochemistry in the SW kidney; ClC-3 was localized in the basal membrane of the collecting ducts and tubules 7 and 30 days after transfer to FW. Renal ClC-3 mRNA expression, however, seemed higher in SW than in FW. The potential role of this chloride channel ClC-3 in osmoregulatory and osmosensing mechanisms is discussed.
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Boudour-Boucheker, N., Boulo, V., Charmantier-Daures, M., Anger, K., Charmantier, G., & Lorin-Nebel, C. (2016). Osmoregulation in larvae and juveniles of two recently separated Macrobrachium species: Expression patterns of ion transporter genes. Comparative Biochemistry and Physiology Part A: Molecular & Integrative Physiology, 195, 39–45.
Résumé: In this comparative study, osmoregulatory mechanisms were analyzed in two closely related species of palaemonid shrimp from Brazil, Macrobrachium pantanalense and Macrobrachium amazonicum. A previous investigation showed that all postembryonic stages of M. pantanalense from inland waters of the Pantanal are able to hyper-osmoregulate in fresh water, while this species was not able to hypo-osmoregulate at high salinities. In M. amazonicum originating from the Amazon estuary, in contrast, all stages are able to hypo-osmoregulate, but only first-stage larvae, late juveniles and adults are able to hyper-osmoregulate in fresh water. The underlying molecular mechanisms of these physiological differences have not been known. We therefore investigated the expression patterns of three ion transporters (NKA α-subunit, VHA B-subunit and NHE3) following differential salinity acclimation in different ontogenetic stages (stage-V larvae, juveniles) of both species. Larval NKAα expression was at both salinities significantly higher in M. pantanalense than in M. amazonicum, whereas no difference was noted in juveniles. VHA was also more expressed in larvae of M. pantanalense than in those of M. amazonicum. When NHE3 expression is compared between the larvae of the two species, further salinity-related differences were observed, with generally higher expression in the inland species. Overall, a high expression of ion pumps in M. pantanalense suggests an evolutionary key role of these transporters in freshwater invasion.
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Boudour-Boucheker, N., Boulo, V., Charmantier-Daures, M., Grousset, E., Anger, K., Charmantier, G., et al. (2014). Differential distribution of V-type H+-ATPase and Na+/K+-ATPase in the branchial chamber of the palaemonid shrimp Macrobrachium amazonicum. Cell and Tissue Research, 357(1), 195–206.
Résumé: V-H+-ATPase and Na+/K+-ATPase were localized in the gills and branchiostegites of M. amazonicum and the effects of salinity on the branchial chamber ultrastructure and on the localization of transporters were investigated. Gills present septal and pillar cells. In freshwater (FW), the apical surface of pillar cells is amplified by extensive evaginations associated with mitochondria. V-H+-ATPase immunofluorescence was localized in the membranes of the apical evaginations and in clustered subapical areas of pillar cells, suggesting labeling of intracellular vesicle membranes. Na+/K+-ATPase labeling was restricted to the septal cells. No difference in immunostaining was recorded for both proteins according to salinity (FW vs. 25 PSU). In the branchiostegite, both V-H+-ATPase and Na+/K+-ATPase immunofluorescence were localized in the same cells of the internal epithelium. Immunogold revealed that V-H+-ATPase was localized in apical evaginations and in electron-dense areas throughout the inner epithelium, while Na+/K+-ATPase occurred densely along the basal infoldings of the cytoplasmic membrane. Our results suggest that morphologically different cell types within the gill lamellae may also be functionally specialized. We propose that, in FW, pillar cells expressing V-H+-ATPase absorb ions (Cl-, Na+) that are transported either directly to the hemolymph space or through a junctional complex to the septal cells, which may be responsible for active Na+ delivery to the hemolymph through Na+/K+-ATPase. This suggests a functional link between septal and pillar cells in osmoregulation. When shrimps are transferred to FW, gill and branchiostegite epithelia undergo ultrastructural changes, most probably resulting from their involvement in osmoregulatory processes.
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Boudour-Boucheker, N., Boulo, V., Lorin-Nebel, C., Elguero, C., Grousset, E., Anger, K., et al. (2013). Adaptation to freshwater in the palaemonid shrimp Macrobrachium amazonicum: comparative ontogeny of osmoregulatory organs. Cell Tissue Res, 353(1), 87–98.
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Dayras, P., Charmantier, G., Chaumot, A., Vigneron, A., Coquery, M., Quéau, H., et al. (2017). Osmoregulatory responses to cadmium in reference and historically metal contaminated Gammarus fossarum (Crustacea, Amphipoda) populations. Chemosphere, 180, 412–422.
Résumé: In order to better understand the variable sensitivities of crustaceans to metals, we investigated the impact of cadmium exposure in 3 populations of Gammarus fossarum from different rivers of France. The first population lives in a Cd-contaminated river from a geochemical background, while the others inhabit Cd-free sites. Osmoregulation, a relevant biomarker to evaluate crustacean health following metal contamination, was used as a proxy to evaluate the intra- and inter-populationnal sensitivities to Cd. Specimens from each population were experimentally exposed to 9 μg Cd2+/L Cd for 7 days and hemolymph osmolality (HO) was then individually measured. In exposed populations, high inter-individual variations in HO values were noted, resulting in their separation into non-impacted and slightly or highly Cd-impacted (with lower HO) animals. In gills of impacted organisms, deep histopathological alterations and protein overexpression of Na+/K+-ATPase and V-H+-ATPase were observed through histology and immunolocalization, while non-impacted animals showed profiles comparable to controls. Moreover, the osmoregulatory processes in the population living in the Cd-contaminated site were impacted by acute Cd exposure in the laboratory as much as for one of the two populations originating from Cd-free sites. The observed changes did not reveal any obvious adaptive osmoregulatory phenomena at the population scale, but they may be due to differences in fitness between individuals and between populations in relation to the features of their respective environments, unrelated with the presence of the metal.
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