Hypo-Osmoregulatory Roles of Vasotocinergic and Isotocinergic Systems in the Intestines of Two European Sea Bass Lineages

European sea bass (Dicentrarchus labrax) are a major aquaculture species that live in habitats with fluctuating salinities that are sometimes higher than in seawater (SW). Atlantic and West- Mediterranean genetic lineages were compared regarding intestinal neuropeptide receptor expression in SW (36%) and following a two-week transfer to hypersalinity (HW, 55%). Phylogenetic analysis revealed seven neuropeptide receptors belonging to the arginine vasotocine (AVTR) family and two isotocin receptors (ITR). Among AVTR paralogs, the highest mRNA levels were recorded for v1a2, with a two- to fourfold upregulation in the European sea bass intestinal sections after transfer of fish toHW. Principal component analysis in posterior intestines showed that v1a2 expression grouped together with the expression and activity of main ion transporters and channels involved in solutecoupled water uptake, indicating a possible role of this receptor in triggering water absorption. v1a1 expression, however, was decreased or did not change after transfer to hypersaline water. Among ITR paralogs, itr1 was the most expressed paralog in the intestine and opposite expression patterns were observed following salinity transfer, comparing intestinal sections. Overall, different expression profiles were observed between genetic lineages for several analyzed genes which could contribute to different osmotic stress-related responses in D. labrax lineages

Intestinal osmoregulatory mechanisms differ in Mediterranean and Atlantic European sea bass: A focus on hypersalinity

European sea bass (Dicentrarchus labrax) migrate towards habitats where salinity can reach levels over 60‰, notably in Mediterranean lagoons. D. labrax are genetically subdivided in Atlantic and Mediterranean lineages and have evolved in slightly different salinities. We compared Atlantic and West-Mediterranean populations regarding their capacity to tolerate hypersalinity with a focus on the involvement of the intestine in solute-driven water reabsorption. Fish were analyzed following a two-week transfer from seawater (SW, 36‰) to either SW or hypersaline water (HW, 55‰). Differences among lineages were observed in posterior intestines of fish maintained in SW regarding NKA activities and mRNA expressions of nkaα1a, aqp8b, aqp1a and aqp1b with systematic higher levels in Mediterranean sea bass. High salinity transfer triggered similar responses in both lineages but at different magnitudes which may indicate slight different physiological strategies between lineages. High salinity transfer did not significantly affect the phenotypic traits measured in the anterior intestine. In the posterior intestine however, the size of enterocytes and NKA activity were higher in HW compared to SW. In this tissue, nka-α1a, nkcc2, aqp8ab and aqp8aa mRNA levels were higher in HW compared to SW as well as relative protein expression of AQP8ab. For aqp1a, 1b, 8aa and 8b, an opposite trend was observed. The sub-apical localization of AQP8ab in enterocytes suggests its role in transepithelial water reabsorption. Strong apical NKCC2/NCC staining indicates an increased Na+ and Cl- reuptake by enterocytes which could contribute to solute-coupled water reuptake in cells where AQP8ab is expressed

Ion uptake pathways in European sea bass Dicentrarchus labrax

Ion uptake mechanisms are diverse in fish species, certainly linked to duplication events that have led to the presence of a multitude of paralogous genes. In fish, Na+ uptake involves several ion transporters expressed in different ionocyte subtypes. In the European sea bass Dicentrarchus labrax, several key transporters potentially involved in Na+ uptake have been investigated in seawater (SW) and following a 2 weeks freshwater (FW) acclimation. Using gel electrophoresis, we have shown that the Na+/H+-exchanger 3 (nhe3, slc9a3) is expressed in gills and kidney at both salinities. Quantitative realtime PCR analysis showed a significantly higher nhe3 expression in fresh water (FW) compared to SW. Its apical localization in a subset of gill ionocytes in freshwater-acclimated fish supports the role of NHE3 in Na+ uptake. Interestingly, NHE3-immunopositive cells also express basolateral Na+/K+/2Cl− cotransporter 1 (NKCC1) and are mainly localized in gill lamella. Among the three nhe2 (slc9a2) paralogs, only nhe2c shows differential branchial expression levels with higher mRNA levels in SW than in FW. The increased branchial expression of the ammonia transporter rhcg1 (Rhesus protein), nhe3 and cytoplasmic carbonic anhydrase (cac) in FW could indicate the presence of a functional coupling between ion transporters to form a Na+/NH4+ exchange complex. Acid-sensing ion channel 4 (asic4) seems not to be expressed in sea bass gills. Na+/Cl- cotransporter (ncc2a or ncc-like) is about three times more expressed in FW compared to SW suggesting coupled Na+ and Cl− uptake in a subset of gill ionocytes. Besides the main pump Na+/K+-ATPase, branchial NCC2a and NHE3 may be key players in ion uptake in sea bass following a long-term freshwater challenge

Inter-individual variability in freshwater tolerance is related to transcript level differences in gill and posterior kidney of European sea bass

Place: Amsterdam Publisher: Elsevier WOS:000526116400015Acclimation to low salinities is a vital physiological challenge for euryhaline fish as the European sea bass Dicentrarchus labrax. This species undertakes seasonal migrations towards lagoons and estuaries where a wide range of salinity variations occur along the year. We have previously reported intraspecific differences in freshwater tolerance, with an average 30% mortality rate. In this study, we bring new evidence of mechanisms underlying freshwater tolerance in sea bass at gill and kidney levels. In fresh water (FW), intraspecific differences in mRNA expression levels of several ion transporters and prolactin receptors were measured. We showed that the branchial Cl-/HCO3- anion transporter (slc26a6c) was over-expressed in freshwater intolerant fish, probably as a compensatory response to low blood chloride levels and potential metabolic alkalosis. Moreover, prolactin receptor a (prlra) and Na+/Cl- cotransporter (ncc1) but not ncc-2a expression seemed to be slightly increased and highly variable between individuals in freshwater intolerant fish. In the posterior kidney, freshwater intolerant fish exhibited differential expression levels of slc26 anion transporters and Na+/K+/2Cl(-) co-transporter 1b (nkcc1b). Lower expression levels of prolactin receptors (prlra, prlrb) were measured in posterior kidney which probably contributes to the failure in ion reuptake at the kidney level. Freshwater intolerance seems to be a consequence of renal failure of ion reabsorption, which is not sufficiently compensated at the branchial level

Tolerance of disease‐vector mosquitoes to brackish water and their osmoregulatory ability

Salinity tolerance is an important trait that governs the ecology of disease‐vector mosquitoes by determining their choice of larval habitat, and consequently their ecological and geographical distribution. Here, we used laboratory strains to determine the osmotic responses of larvae of obligate freshwater disease‐vector mosquitoes (Aedes aegypti, Aedes albopictus, Anopheles coluzzii, An. gambiae, Culex pipiens, and Cx. quinquefasciatus) and assessed their relationship with salinity tolerance. First, we analyzed the acute dose–mortality response of fourth‐instar larvae to salinity; then, we measured their hemolymph osmolality after 24‐h exposure to varying salinities. We found that Ae. albopictus was the most tolerant species, followed by An. coluzzii, Ae. aegypti, Cx. quinquefasciatus, and An. gambiae, in decreasing order. Cx. pipiens was the least tolerant species. All mosquitoes were hyper‐iso‐osmoregulators, but with species‐specific differences. Specifically, hemolymph osmolality in deionized water varied among species, and Cx. pipiens and the two Aedes species showed the lowest and highest osmolality. Although all species were osmoconformers at higher salinity values, hemolymph osmolality approached environmental osmolality more rapidly in species of the Culex genus, compared with Aedes species where it increased slowly. Moreover, hemolymph osmolality in deionized water was significantly correlated with tolerance to salinity across species. This could allow predicting the salinity tolerance of untested species on the basis of their osmoregulatory ability. However, this correlation disappeared when considering the hemolymph osmolality of larvae exposed to salinities higher than deionized water

Effects of copper and parasitism on osmoregulation in the European eel Anguilla anguilla

International audienc

Salinity stress from the perspective of the energy-redox axis: Lessons from a marine intertidal flatworm

In the context of global change, there is an urgent need for researchers in conservation physiology to understand the physiological mechanisms leading to the acquisition of stress acclimation phenotypes. Intertidal organisms continuously cope with drastic changes in their environmental conditions, making them outstanding models for the study of physiological acclimation. As the implementation of such processes usually comes at a high bioenergetic cost, a mitochondrial/oxidative stress approach emerges as the most relevant approach when seeking to analyze whole-animal responses. Here we use the intertidal flatworm Macrostomum lignano to analyze the bioenergetics of salinity acclimation and its consequences in terms of reactive oxygen/nitrogen species formation and physiological response to counteract redox imbalance. Measures of water fluxes and body volume suggest that M. lignano is a hyper-/iso-regulator. Higher salinities were revealed to be the most energetically expensive conditions, with an increase in mitochondrial density accompanied by increased respiration rates. Such modifications came at the price of enhanced superoxide anion production, likely associated with a high caspase 3 upregulation. These animals nevertheless managed to live at high levels of environmental salinity through the upregulation of several mitochondrial antioxidant enzymes such as superoxide dismutase. Contrarily, animals at low salinities decreased their respiration rates, reduced their activity and increased nitric oxide formation, suggesting a certain degree of metabolic arrest. A contradictory increase in dichlorofluorescein fluorescence and an upregulation of gluthathione-S-transferase pi 1 (GSTP1) expression were observed in these individuals. If animals at low salinity are indeed facing metabolic depression, the return to seawater may result in an oxidative burst. We hypothesize that this increase in GSTP1 could be a “preparation for oxidative stress”, i.e. a mechanism to counteract the production of free radicals upon returning to seawater. The results of the present study shed new light on how tolerant organisms carry out subcellular adaptations to withstand environmental change

Effect of salinity and temperature on the expression of genes involved in branchial ion transport processes in European sea bass

The responses of European sea bass to temperature increase and salinity decrease were investigated measuring mRNA expression levels of main genes involved in ion transport. Juvenile fish were pre-acclimated to seawater (SW) at 18 °C (temperate) or 24 °C (warm) for two weeks and then transferred for two weeks to either fresh water (FW) or SW at the respective temperature. Unlike temperate conditions, there is no change in Na+/K+-ATPase α1a (nka α1a) and Na+/H+ exchanger 3 (nhe3) mRNA expression following FW transfer in warm conditions. This is linked to the high expression of these genes in warm SW compared to temperate SW. Na+/Cl−-cotransporter (ncc2a) expression however is increased following FW transfer in temperate and warm conditions. Main transporters involved in ion excretion (Na+/K+/2Cl--1 cotransporter, nkcc1 and cystic fibrosis transmembrane conductance regulator, cftr) as well as nitrogen excretion (Rh-glycoproteins, rhcg1 and rhbg) and acid-base regulation (V-H+-ATPase, vha-a and b) are highly expressed in SW warm conditions vs FW warm. Overall, our results suggest a higher activation of ion transport processes in warm conditions and more strikingly in SW. This is linked to a strong interplay between diverse ion transporters in order to coordinate physiological responses at the gill level

The role of salinity on genome‐wide DNA methylation dynamics in European sea bass gills

Epigenetic modifications, like DNA methylation, generate phenotypic diversity in fish and ultimately lead to adaptive evolutionary processes. Euryhaline marine species that migrate between salinity-contrasted habitats have received little attention regarding the role of salinity on whole-genome DNA methylation. Investigation of salinity-induced DNA methylation in fish will help to better understand the potential role of this process in salinity acclimation. Using whole-genome bisulfite sequencing, we compared DNA methylation patterns in European sea bass (Dicentrarchus labrax) juveniles in seawater and after freshwater transfer. We targeted the gill as a crucial organ involved in plastic responses to environmental changes. To investigate the function of DNA methylation in gills, we performed RNAseq and assessed DNA methylome-transcriptome correlations. We showed a negative correlation between gene expression levels and DNA methylation levels in promoters, first introns and first exons. A significant effect of salinity on DNA methylation dynamics with an overall DNA hypomethylation in freshwater-transferred fish compared to seawater controls was demonstrated. This suggests a role of DNA methylation changes in salinity acclimation. Genes involved in key functions as metabolism, ion transport and transepithelial permeability (junctional complexes) were differentially methylated and expressed between salinity conditions. Expression of genes involved in mitochondrial metabolism (tricarboxylic acid cycle) was increased, whereas the expression of DNA methyltransferases 3a was repressed. This study reveals novel links between DNA methylation, mainly in promoters and first exons/introns, and gene expression patterns following salinity change