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To understand the response of freshwater organisms to rising environmental salinity, it is essential to investigate their osmoionic regulatory physiology. Our laboratory experiment investigated the transfer of Palaemon argentinus (Nobili, 1901) from 2[per thousand] (control condition) to concentrated salinity (15 and 25[per thousand]) for short- (6 h), medium- (48 h), and long-term (>504 h) acclimation periods. We measured relevant parameters in the shrimp's haemolymph, the time course of the response of branchial V-[H.sup.+]-ATPase (VHA), [Na.sup.+], [K.sup.+]--ATPase (NKA), carbonic anhydrase (CA) activity, and muscle water content. Upon prolonged acclimation to 15[per thousand] (hyper-regulating condition), shrimp reached a new steady state of haemolymph osmolality by tightly adjusting ion concentrations to levels higher than the external medium. While NKA and CA activities recovered their pre-transfer levels, the downregulation of VHA suggests other functions rather than ion uptake after prolonged acclimation to 15[per thousand]. The activity of the three transport-related enzymes remained almost unchanged at the highest salinity (isosmotic condition), leading to increasing osmotic pressure and ion concentration after prolonged acclimation to 25[per thousand]. Although the freshwater shrimp studied here retains a certain degree of tolerance to high salinity, a common trait in palaemonid shrimps, our results highlight that 25[per thousand] represents a significant hypertonic challenge for this species. Keywords: carbonic anhydrase, gills, ionic regulation, [Na.sup.+], [K.sup.+]--ATPase, Palaemon argentinus, (V)-type H+--ATPase, Introduction Inland aquatic ecosystems from southern regions in South America as the ecoregion of the Pampas (Argentina), are vulnerable to increasing salinization due to human activities and global climate change [...]

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. [ABSTRACT FROM AUTHOR]

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 to HW. 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 solute-coupled 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. [ABSTRACT FROM AUTHOR]

Life in fresh water is osmotically and energetically challenging for living organisms, requiring increases in ion uptake from dilute environments. However, mechanisms of ion uptake from freshwater environments are still poorly understood and controversial, especially in arthropods, for which several hypothetical models have been proposed based on incomplete data. One compelling model involves the proton pump V-type H+ ATPase (VHA), which energizes the apical membrane, enabling the uptake of Na+ (and other cations) via an unknown Na+ transporter (referred to as the “Wieczorek Exchanger” in insects). What evidence exists for this model of ion uptake and what is this mystery exchanger or channel that cooperates with VHA? We present results from studies that explore this question in crustaceans, insects, and teleost fish. We argue that the Na+/H+ antiporter (NHA) is a likely candidate for the Wieczorek Exchanger in many crustaceans and insects; although, there is no evidence that this is the case for fish. NHA was discovered relatively recently in animals and its functions have not been well characterized. Teleost fish exhibit redundancy of Na+ uptake pathways at the gill level, performed by different ion transporter paralogs in diverse cell types, apparently enabling tolerance of low environmental salinity and various pH levels. We argue that much more research is needed on overall mechanisms of ion uptake from freshwater habitats, especially on NHA and other potential Wieczorek Exchangers. Such insights gained would contribute greatly to our general understanding of ionic regulation in diverse species across habitats. [ABSTRACT FROM AUTHOR]

1,4,5-trisphosphate (I[P.sub.3])-dependent [Ca.sup.2+] signaling regulates gonad function, fertility, and rhythmic posterior body wall muscle contraction (pBoc) required for defecation in Caenorhabditis elegans. Store-operated [Ca.sup.2+] entry (SOCE) is activated during endoplasmic reticulum (ER) [Ca.sup.2+] store depletion and is believed to be an essential and ubiquitous component of [Ca.sup.2+] signaling pathways. SOCE is thought to function to refill [Ca.sup.2+] stores and modulate [Ca.sup.2+] signals. Recently, stromal interaction molecule 1 (STIM1) was identified as a putative ER [Ca.sup.2+] sensor that regulates SOCE. We cloned a full-length C. elegans stim-1 cDNA that encodes a 530--amino acid protein with ~21% sequence identity to human STIM1. Green fluorescent protein (GFP)--tagged STIM-1 is expressed in the intestine, gonad sheath cells, and spermatheca. Knockdown of stim-1 expression by RNA interference (RNAi) causes sterility due to loss of sheath cell and spermatheca contractile activity required for ovulation. Transgenic worms expressing a STIM-1 EF-hand mutant that constitutively activates SOCE in Drosophila and mammalian cells are sterile and exhibit severe pBoc arrhythmia, stim-1 RNAi dramatically reduces STIM-1::GFP expression, suppresses the EF-hand mutation--induced pBoc arrhythmia, and inhibits intestinal store-operated [Ca.sup.2+] (SOC) channels. However, stim-1 RNAi surprisingly has no effect on pBoc rhythm, which is controlled by intestinal oscillatory [Ca.sup.2+] signaling, in wild type and I[P.sub.3] signaling mutant worms, and has no effect on intestinal [Ca.sup.2+] oscillations and waves. Depletion of intestinal [Ca.sup.2+] stores by RNAi knockdown of the ER [Ca.sup.2+] pump triggers the ER unfolded protein response (UPR). In contrast, stim-1 RNAi fails to induce the UPR. Our studies provide the first detailed characterization of STIM-1 function in an intact animal and suggest that SOCE is not essential for certain oscillatory [Ca.sup.2+] signaling processes and for maintenance of store [Ca.sup.2+] levels in C. elegans. These findings raise interesting and important questions regarding the function of SOCE and SOC channels under normal and pathophysiological conditions.

The copepod Eurytemora affinis has an unusually broad salinity range, as some populations have recently invaded freshwater habitats independently from their ancestral saline habitats. Prior studies have shown evolutionary shifts in ion transporter activity during freshwater invasions and localization of ion transporters in newly discovered "Crusalis organs" in the swimming legs. The goals of this study were to localize and quantify expression of ion transport enzymes V-type H+-ATPase (VHA) and Na+/K+-ATPase (NKA) in the swimming legs of E. affinis and determine the degree of involvement of each leg in ionic regulation. We confirmed the presence of two distinct types of ionocytes in the Crusalis organs. Both cell types expressed VHA and NKA, and in the freshwater population the location of VHA and NKA in ionocytes was, respectively, apical and basal. Quantification of in situ expression of NKA and VHA established the predominance of swimming leg pairs 3 and 4 in ion transport in both saline and freshwater populations. Increases in VHA expression in swimming legs 3 and 4 of the freshwater population (in fresh water) relative to the saline population (at 15 PSU) arose from an increase in the abundance of VHA per cell rather than an increase in the number of ionocytes. This result suggests a simple mechanism for increasing ion uptake in fresh water. In contrast, the decline in NKA expression in the freshwater population arose from a decrease in ionocyte area in legs 4, likely resulting from decreases in number or size of ionocytes containing NKA. Such results provide insights into mechanisms of ionic regulation for this species, with added insights into evolutionary mechanisms underlying physiological adaptation during habitat invasions. [ABSTRACT FROM AUTHOR]

This study combines a cellular and molecular analysis of the Na+/K+/2CI- cotransporter (NKCC) to determine the osmoregulatory role of this protein in different tissues during the ontogeny of the sea bass. We have characterized the complete sequence of the NKCC1 isoform isolated from the sea bass gills and have identified, by immunofluorescence, NKCC1, and other isoforms, within the epithelium of the major osmoregulatory organs. Different (absorptive and secretory) functions have been attributed to this protein according to the tissue and salinity. The effects of short- (1–4 days), medium- (7–21 days) and long (6 months)-term freshwater (FW) adaptations were investigated, in comparison with seawater (SW)-maintained sea bass. In adult sea bass after long-term adaptation to FW and SW, the gills had the highest expression of NKCC mRNA compared with the median/posterior kidney and to the posterior intestine. Expression of NKCC mRNA in the kidney was 95% (SW) and 63% (FW) lower, and in the intestine 98% (SW) and 77% (FW) lower. Compared to SW-maintained sea bass, long-term FW adaptation induced a significant 5.6-fold decrease in the branchial NKCC gene expression whereas the intestinal and renal expressions did not vary significantly. The cells of the intestine and collecting ducts as well as a part of the epithelium lining the urinary bladder expressed NKCC apically. Within the gill chloride cells, NKCC was found basolaterally in SW-acclimated fish; some apically stained cells were detected after 7 days of FW exposure and their relative number increased progressively following FW acclimation. The appearance of FW-type chloride cells induces a functional shift of the gills from a secretory to an absorptive epithelium, which was only completed after long-term exposure to FW. Short- and medium-term exposure to FW induced a progressive decrease in total NKCC content and an increase in functionally different branchial chloride cells. During development, the cotransporter was already expressed in tegumentary ionocytes and along the digestive tract of late embryos. NKCC was recorded in the branchial chamber and along the renal collecting ducts in prelarvae and also in the dorsal part of the urinary bladder in larvae. The expression of NKCC along the osmoregulatory epithelial cells and the presence of Na+/K+-ATPase within these cells contribute to the increase of the osmoregulatory capacity during sea bass ontogeny. [ABSTRACT FROM AUTHOR]

Abstract 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. Highlights • Transcript levels of ion transporters were investigated in Dicentrarchus labrax gills. • Key branchial transporters involved in freshwater acclimation were identified. • A focus was made on the expression of Na+/H+-exchanger (NHE) paralogs. • The Na+/Cl- - cotransporter (NCC2a) was identified for the first time in sea bass. • Different ionocytes subtypes were compared in filaments vs lamella [ABSTRACT FROM AUTHOR]

1,4,5-trisphosphate (1P3)-dependent Ca2+ signaling regulates gonad function, fertility, and rhythmic posterior body wall muscle contraction (pBoc) required for defecation in Caenorhabditis elegans. Store-operated Ca2+ entry (SOCE) is activated during endoplasmic reticulum (ER) Ca2+ store depletion and is believed to be an essential and ubiquitous component of Ca2+ signaling pathways. SOCE is thought to function to refill Ca2+ stores and modulate Ca2+ signals. Recently, stromal interaction molecule 1 (STIM1) was identified as a putative ER Ca2+ sensor that regulates SOCE. We cloned a full-length C. elegans stim-1 cDNA that encodes a 530-amino acid protein with ∼21% sequence identity to human STIM1. Green fluorescent protein (GFP)-tagged STIM-1 is expressed in the intestine, gonad sheath cells, and spermatheca. Knockdown of stim-1 expression by RNA interference (RNAi) causes sterility due to loss of sheath cell and spermatheca contractile activity required for ovulation. Transgenic worms expressing a STIM-1 EF-hand mutant that constitutively activates SOCE in Drosophila and mammalian cells are sterile and exhibit severe pBoc arrhythmia. stim-1 RNAi dramatically reduces STIM-1::GFP expression, suppresses the EF-hand mutation-induced pBoc arrhythmia, and inhibits intestinal store-operated Ca2+ (SOC) channels. However, stim-1 RNAi surprisingly has no effect on pBoc rhythm, which is controlled by intestinal oscillatory Ca2+ signaling, in wild type and 1P3 signaling mutant worms, and has no effect on intestinal Ca2+ oscillations and waves. Depletion of intestinal Ca2+ stores by RNAi knockdown of the ER Ca2+ pump triggers the ER unfolded protein response (UPR). In contrast, stim-1 RNAi fails to induce the UPR. Our studies provide the first detailed characterization of STIM-1 function in an intact animal and suggest that SOCE is not essential for certain oscillatory Ca2+ signaling processes and for maintenance of store Ca2+ levels in C. elegans. These findings raise interesting and important questions regarding the function of SOCE and SOC channels under normal and pathophysiological conditions. [ABSTRACT FROM AUTHOR]

Abstract To examine the physiological roles of arginine vasotocin receptor (AVTR) and isotocin receptor (ITR) in osmoregulation of a euryhaline teleost, the marbled eel (Anguilla marmorata), three different genes coding for AVTRV1a2, AVTRV2 and ITR were cloned by screening an A. marmorata cDNA library. These receptors were expressed differentially and ubiquitously in the eight tissues we examined. The changes in mRNA expression levels of AVTRV1a2, AVTRV2, and ITR were assessed in a time-course study following salinity transfer from fresh water (FW, 0‰) to fresh water (FW, 0‰), brackish water (BW, 10‰) or saline water (SW, 25‰). When eels were transferred to BW, mRNA levels underwent an adaptive period, from 0 to 24 h, and a chronic regulatory period, starting at 24 h after transfer. In the adaptive period, the relative mRNA expression of AVTRV1a2, AVTRV2, and ITR increased in BW. But after this adaptive period, the mRNA levels of the three genes were significantly decreased compared to FW (control group, 0 h). The mRNA expression levels of AVTRV1a2, AVTRV2 and ITR were low in SW. The protein level of AVTRV1a2, a key protein in the brain, was also investigated and found to be consistent with mRNA results. Our results indicated that the nonapeptide receptor system may play a role in the acute stress response induced by hyper-osmotic challenge in marbled eels. Highlights • Full-length cDNAs encoding AVTRV1a2, AVTRV2 and ITR were cloned in A. marmorata. • These receptors were expressed ubiquitously in eight examined tissues. • The mRNA and protein temporal expressions were stated in salinity acclimation. [ABSTRACT FROM AUTHOR]

• Freshwater intolerance is not due to an altered ion uptake at the gills. • Cl− uptake-related genes are less expressed in the kidney of freshwater (FW) intolerant fish vs FW tolerant fish. • Branchial and renal prolactin receptors are differentially expressed between freshwater tolerance phenotypes. Acclimation 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−/HCO 3 − 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− cotransporter 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. [ABSTRACT FROM AUTHOR]

Life in fresh water is osmotically and energetically challenging for living organisms, requiring increases in ion uptake from dilute environments. However, mechanisms of ion uptake from freshwater environments are still poorly understood and controversial, especially in arthropods, for which several hypothetical models have been proposed based on incomplete data. One compelling model involves the proton pump V-type H + ATPase (VHA), which energizes the apical membrane, enabling the uptake of Na + (and other cations) via an unknown Na + transporter (referred to as the "Wieczorek Exchanger" in insects). What evidence exists for this model of ion uptake and what is this mystery exchanger or channel that cooperates with VHA? We present results from studies that explore this question in crustaceans, insects, and teleost fish. We argue that the Na + /H + antiporter (NHA) is a likely candidate for the Wieczorek Exchanger in many crustaceans and insects; although, there is no evidence that this is the case for fish. NHA was discovered relatively recently in animals and its functions have not been well characterized. Teleost fish exhibit redundancy of Na + uptake pathways at the gill level, performed by different ion transporter paralogs in diverse cell types, apparently enabling tolerance of low environmental salinity and various pH levels. We argue that much more research is needed on overall mechanisms of ion uptake from freshwater habitats, especially on NHA and other potential Wieczorek Exchangers. Such insights gained would contribute greatly to our general understanding of ionic regulation in diverse species across habitats., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Lee, Charmantier and Lorin-Nebel.)