Research paperInter-individual variability in freshwater tolerance is related to transcript level differences in gill and posterior kidney of European sea bass
Introduction
In transitional habitat such as lagoons, fish have to deal with a wide range of changing environmental parameters and therefore a high phenotypic plasticity is beneficial to cope with fluctuating environments. Salinity can rapidly drop in these habitats through freshwater (FW) supplies by rainfalls or rivers. Low-salinity environments can lead to differential distributions of marine species according to their acclimation capacity (Pierce et al., 2012, Wong et al., 1999). Differential habitat distribution has also been observed between individuals within the same species as in stickleback Gasterosteus aculateus and in mummichog, Fundulus heteroclitus, showing a differential capacity to regulate ions in FW (Scott et al., 2004a, Scott et al., 2004b, McCairns and Bernatchez, 2009). To maintain hydromineral balance in FW environments, fish have to minimise diffusive ion losses and compensate them by actively (re)absorbing ions at the gill and posterior kidney levels (Hickman and Trump, 1969, Dantzler, 1996). The European sea bass, Dicentrarchus labrax, is known to undertake seasonal migrations in transitional habitats where salinity fluctuates (Kelley, 1988, Barnabé, 1989, Waldman, 1995). Previous studies highlighted intraspecific differences in FW tolerance in this species at different ages (Giffard-Mena et al., 2008, L’Honoré et al., 2019, Nebel et al., 2005) with about 30% mortality following FW exposure. Fish that did not tolerate FW could survive when they were rechallenged to SW conditions and no intraspecific variation was observed in SW (Giffard-Mena et al., 2008, L’Honoré et al., 2019). In F. heteroclitus, Scott et al., 2004a, Scott et al., 2004b concluded that the divergence in osmoregulatory capacities may result in different capacities to absorb and reabsorb Na+ and Cl− at gill and/or kidney levels. In Nebel et al. (2005), it has been suggested that the kidney might be responsible for the osmoregulatory failure detected in FW intolerant fish, linked to a low renal Na+-K+-ATPase (NKA) activity and a lower kidney tubular density. In L’Honoré et al. (2019), authors highlighted that intraspecific variation in FW tolerance of European sea bass is supported by strong differences in nka α1a expression in the posterior kidney while no difference was measured at the gill level. Authors also showed differences in corticosteroid receptors mRNA levels (gr1, gr2 and mr) with lower expression levels in FW intolerant fish compared to FW tolerant. This suggests an impaired hormonal and stress regulation between both FW-tolerance phenotypes. Pituitary prolactin (PRL) is a key hormone involved in FW acclimation by promoting the maintenance of the hydromineral balance (Hirano, 1986; Manzon, 2002, Sakamoto and McCormick, 2006, Breves et al., 2014, Bossus et al., 2017). PRL interacts with PRL receptor (PRLR) to regulate Na+ efflux, water permeability and the differentiation of ionocytes expressing Na+/Cl− cotransporter (NCC) as shown in tilapia and zebrafish (Breves et al., 2013, Breves et al., 2010, Dharmamba et al., 1967, Dharmamba and Maetz, 1972). Prolactin receptors are cell surface receptors known to be expressed in osmoregulatory organs of many teleost species (Manzon, 2002). As for many fish species like Takifugu rubripes, two paralogs of prlr were identified in D. labrax genome called prlra and prlrb (Lee et al., 2006, Tine et al., 2014). It remains to be determined if both prlr paralogs display the same expression pattern according to salinity and if mRNA levels differ between FW-tolerance phenotypes in European sea bass.
In fish, blood pH levels are salinity-dependent with lower blood pH at low salinity than in SW as it was demonstrated in European sea bass by Shrivastava et al. (2019). In 8 month-old European sea bass, FW intolerance was characterised by a lower blood osmolality associated with an elevated Na+/Cl− ratio, indicating a metabolic alkalosis (L’Honoré et al., 2019). To regulate a high blood pH, fish have to excrete bases as HCO3−, mainly associated with Cl− uptake (Maetz and García Romeu, 1964, Goss and Wood, 1990, Goss and Wood, 1991, Tresguerres et al., 2006). The apical anion transporters SLC26A6, involved in Cl−/HCO3− and Cl−/oxalate exchanges, are known to be widespread among species in osmoregulatory organs including the posterior kidney (Mount and Romero, 2004, Sardella and Brauner, 2007, Xie et al., 2002, Knauf et al., 2018) and gills (Perry et al., 2009, Boyle et al., 2015) and could be potential entry routes for HCO3− in intolerant European sea bass to FW. In the gulf toadfish for example, slc26a6 was highly expressed in kidney (Grosell et al., 2009) to reabsorb Cl− from the lumen to the blood. Among three different SLC26 anion transporters in zebrafish (SLC26A6, SLC26A3 and SLC26A4, called za6, za3 and za4), za6 seemed to be the most expressed in gills and was overexpressed when fish were transferred to water with low Cl− or NaHCO3 (Perry et al., 2009). Moreover, gene knockdown of slc26a6c resulted in a reduction in Cl− uptake in zebrafish larvae confirming the major role of this transporter in Cl− uptake (Bayaa et al., 2009, Perry et al., 2009). Guh et al. (2015) localised SLC26 genes apically in gill ionocytes, called SLC26 cells, but to our knowledge nothing is known about SLC26 localisation in the fish kidney. In D. labrax, the only record about SLC26A6 (most probably SLC26A6c) is very recent and shows a high mRNA expression in the anterior intestine to promote Cl− transepithelial transport to the blood and HCO3− excretion (Alves et al., 2019).
The basolateral Na+/K+-ATPase (NKA) (Hwang et al., 2011, Kumai and Perry, 2012) is a key player in Na+ and Cl− uptake and its importance in FW acclimation has been demonstrated in many fish species (Hiroi et al., 2008, Inokuchi et al., 2008, Watanabe et al., 2008, Hsu et al., 2014, Bollinger et al., 2016). Nka α1a transcriptional expression in the posterior kidney was previously shown to be related to FW tolerance in European sea bass (L’Honoré et al., 2019). One other key cotransporter known for Na+ and Cl− uptake in fish is the NCC-2A or NCC-like (SLC12A10 or SLC12A10.2), that plays a crucial role in FW acclimation in many fish species including European sea bass (Inokuchi et al., 2008, Wang et al., 2009, Hwang et al., 2011, Blondeau-Bidet et al., 2019). SLC12A10 is localised apically in FW-type branchial ionocytes (i.e. NCC-type cells) in several species (Hiroi et al., 2008, Inokuchi et al., 2008, Guh et al., 2015, Blondeau-Bidet et al., 2019). It is highly expressed in FW compared to SW fish gills (Hiroi et al., 2008, Inokuchi et al., 2008, Wang et al., 2009, Blondeau-Bidet et al., 2019). In posterior kidney, slc12a10.1 paralog has been shown to be highly expressed in zebrafish and in Mozambique tilapia gills compared to other organs (Hiroi et al., 2008, Wang et al., 2009). The Na+/H+ exchanger-3 (NHE3) is also localised in the apical membrane of another ionocyte subtype at the gill level (Inokuchi et al., 2008, Watanabe et al., 2008, Hwang et al., 2011, Blondeau-Bidet et al., 2019) and functionally coupled to several other ion transporters facilitating Na+ uptake (Dymowska et al., 2015). Anion exchanger 1 (AE1) is mainly known to play a role in bicarbonate transport to the blood and Cl− secretion at the gill and kidney (mammal medullary collecting duct cells) levels (Barone et al., 2004, Lee et al., 2011). AE1 is thought to be functionally linked to apical V-H+-ATPase (VHA) in order to complete acid secretion. At the gill level, AE1 is localised basolaterally in zebrafish HR cells (Lee et al., 2011). Its role was investigated in medaka Oryzias latipes and in zebrafish gills (Lee et al., 2011, Hsu et al., 2014, Liu et al., 2016). In both species, the two analysed paralogs (ae1a and ae1b) were expressed in gills with ae1b being over-expressed in a low-Na+ environment. It has also been suggested that AE1 is involved in Cl− absorption and HCO3− secretion (Evans et al., 2005, Hwang and Lee, 2007, Hwang and Perry, 2010) but its localisation in basolateral membranes of ionocytes in pufferfish Tetraodon nigroviridis and in milkfish Chanos chanos seems not in favour with this hypothesis (Tang and Lee, 2007, Tang et al., 2011). In European sea bass, no data is available on VHA and AE1 localization. VHA mRNA expression and protein activity measurements have shown the presence of VHA in D. labrax gills in FW media (Sinha et al., 2015, Blondeau-Bidet et al., 2019).
Regarding Na+/K+/2Cl− cotransporters (NKCC), three paralogs have been described in teleosts: basolateral NKCC1a mainly expressed in the gills and NKCC1b, both dedicated to NaCl secretion, and the apical NKCC2 mainly expressed in the kidney and intestine and attributed to NaCl reabsorption (Haas and Forbush, 2000, Teranishi et al., 2013). NKCC1 paralogs are expressed in SW-type ionocytes in numerous teleost species including the European sea bass (Lorin-Nebel et al., 2006, Inokuchi et al., 2008, Buhariwalla et al., 2012, Breves et al., 2014). NKCC2 was detected in tilapia and in D. labrax intestine (Hiroi et al., 2008, Alves et al., 2019), but there is no data available on its localisation in the gills and in the posterior kidney of D. labrax.
European sea bass exhibiting a FW intolerant phenotype face a severe hydromineral imbalance and we will analyse if this failure originates from a failure in ion uptake at the kidney and/or gill levels. The involvement of the gill in differential ion uptake capacity of D. labrax will be analysed by comparing the expression profile of the main ion transporters in tolerant and intolerant fish to FW. Little information is available about ion transporter expression at the kidney level. This is a first tentative to identify key renal ion transporters in FW kidney and to detect intraspecific differences in the transcriptional profile between tolerant and intolerant fish to FW.
Section snippets
Tissue sampling
European sea bass juveniles (N = 350) were reared at Ifremer Station at Palavas-les-flots (Hérault, France) in recirculating SW (osmolality: 1208 mOsm kg−1, Na+: 515 mmol L−1, Cl−: 737 mmol L−1) under a 12/12 h light/dark photoperiod at 20 °C. At the age of 8 months (13.59 ± 0.12 cm, 32.19 ± 2.62 g), 300 fish were then transferred to brackish water (BW; osmolality: 475 mOsm kg−1) for 24 h and then transferred to dechlorinated tap FW (osmolality: 8 mOsm kg−1, Na+: 2 mmol L−1, Cl−: 3.5 mmol L−1)
RNA extraction and complementary DNA (cDNA) synthesis
Tissues were thawed on ice in lysis buffer using the total RNA extraction kit (Nucleospin® RNA, Macherey-Nagel, Germany) before performing the extraction. Quantity and purity (A260/280 ratio) of extracted RNA were verified using a spectrophotometer (NanoDrop™ One/OneC Spectrophotometer, Thermo Scientific, Waltham, MA, USA). One microgram of RNA was used to generate the complementary DNA (cDNA) using the qScript™ cDNA SuperMix (Quanta Biosciences™) providing all necessary components for
Results
Among the 300 fish challenged in FW, 28% of them were detected and characterised as FW intolerant fish and 70% as FW tolerant fish.
Discussion
The comparative analysis of key genes and proteins involved in the maintenance of hydromineral balance in fish exhibiting different capacities to tolerate FW is a powerful tool to investigate intraspecific variation in FW tolerance in euryhaline species. In FW, fish have to minimise ion loss and compensate through active ion uptake occurring at interfaces with their surroundings. Most studies investigating FW osmoregulation focused at the gill level in adults or at integument level in larvae
Conclusion
This study is the first to highlight that freshwater intolerance in European sea bass is linked with lower mRNA expression of slc26a6 genes involved in Cl− uptake in the posterior kidney. Regarding prolactin receptors, we showed a differential endocrine control in FW between the tolerant and intolerant European sea bass associated to the incapacity to maintain blood hydromineral balance in FWi sea bass. Facing strong rainfalls during their migration to transitional habitats like lagoons,
CRediT authorship contribution statement
L’Honoré Thibaut: Formal analysis, Investigation, Visualization, Writing - original draft, Writing - review & editing. Farcy Emilie: Conceptualization, Methodology, Validation, Writing - review & editing, Resources, Supervision. Blondeau-Bidet Eva: Methodology, Validation, Formal analysis, Visualization. Lorin-Nebel Catherine: Conceptualization, Methodology, Validation, Writing - review & editing, Resources, Funding acquisition, Supervision.
Declaration of Competing Interest
The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.
Acknowledgments
We would like to thank Philippe Clair from the qPCR CEMEB platform for his help, and François Ruelle, Marie-Odile Blanc, Frederic Clota, Stephane Lallement and Alain Vergnet from Ifremer Palavas-les-flots for the maintenance of the fish. This research was partially granted from CeMEB (Centre Méditerranée en Environnement et Biodiversité) labex exploratory projects.
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