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Since 2000, a well-established population of the invasive oriental shrimp Palaemon macrodactylus has been present in fully marine conditions in the southwestern Atlantic Ocean (~38° S). To assess the physiological performance of this atypical population restricted to fully marine conditions, we conducted a laboratory experiment in which individuals were transferred from 35 ‰S (local seawater) to 2 ‰S; 5 ‰S; 10 ‰S; 20 ‰S; 50 ‰S and 60‰ for short (6 h), medium (48 h), and long (>504 h) acclimation periods. We measured the time course response of relevant parameters in the shrimp's hemolymph; activity of Na+, K+-ATPase (NKA), and V-H+-ATPase (VHA); and muscle water content. Shrimp showed great osmoregulatory plasticity, being able to survive for long periods between 5 ‰S and 50 ‰S, whereas no individual survived after transfer to either 2 ‰S or 60 ‰S. Shrimp hyper-regulated hemolymph osmolality at 5 ‰S and 10 ‰S, hypo-regulated at 35 ‰S and 50 ‰S, and isosmoticity was close to 20 ‰S. Compared to 35 ‰S, prolonged acclimation to 5 ‰S caused a decrease in hemolymph osmolality (~34%) along with sodium and chloride concentrations (~24%); the NKA and VHA activities decreased by ~52% and ~88%, respectively, while muscle water content was tightly regulated. Our results showed that the atypical population of P. macrodactylus studied here lives in a chronic hypo-osmo-ion regulatory state and suggest that fully marine conditions contribute to its poor performance at the lower limit of salinity tolerance (<5 ‰S).

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Mesembryanthemum crystallinum (common ice plant), a facultative CAM plant, shifts from C3 to CAM photosynthesis under salt stress, enhancing water use efficiency. Here we used transcriptomics, proteomics, and targeted metabolomics to profile molecular changes during the diel cycle of C3 to CAM transition. The results confirmed expected changes associated with CAM photosynthesis, starch biosynthesis and degradation, and glycolysis/gluconeogenesis. Importantly, they yielded new discoveries: 1) Transcripts displayed greater circadian regulation than proteins. 2) Oxidative phosphorylation and inositol methylation may play important roles in initiating the transition. 3) V-type H+-ATPases showed consistent transcriptional regulation, aiding in vacuolar malate uptake. 4) A protein phosphatase 2C, a major component in the ABA signaling pathway, may trigger the C3 to CAM transition. Our work highlights the potential molecular switches in the C3 to CAM transition, including the potential role of ABA signaling. SIGNIFICANCE: The common ice plant is a model facultative CAM plant, and under stress conditions it can shift from C3 to CAM photosynthesis within a three-day period. However, knowledge about the molecular changes during the transition and the molecular switches enabling the transition is lacking. Multi-omic analyses not only revealed the molecular changes during the transition, but also highlighted the importance of ABA signaling, inositol methylation, V-type H+-ATPase in initiating the shift. The findings may explain physiological changes and nocturnal stomatal opening, and inform future synthetic biology effort in improving crop water use efficiency and stress resilience.

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Diatoms, dinoflagellates, and coccolithophores are dominant groups of marine eukaryotic phytoplankton that are collectively responsible for the majority of primary production in the ocean.1 These phytoplankton contain additional intracellular membranes around their chloroplasts, which are derived from ancestral engulfment of red microalgae by unicellular heterotrophic eukaryotes that led to secondary and tertiary endosymbiosis.2 However, the selectable evolutionary advantage of these membranes and the physiological significance for extant phytoplankton remain poorly understood. Since intracellular digestive vacuoles are ubiquitously acidified by V-type H+-ATPase (VHA),3 proton pumps were proposed to acidify the microenvironment around secondary chloroplasts to promote the dehydration of dissolved inorganic carbon (DIC) into CO2, thus enhancing photosynthesis.4,5 We report that VHA is localized around the chloroplasts of centric diatoms and that VHA significantly contributes to their photosynthesis across a wide range of oceanic irradiances. Similar results in a pennate diatom, dinoflagellate, and coccolithophore, but not green or red microalgae, imply the co-option of phagocytic VHA activity into a carbon-concentrating mechanism (CCM) is common to secondary endosymbiotic phytoplankton. Furthermore, analogous mechanisms in extant photosymbiotic marine invertebrates6,7,8 provide functional evidence for an adaptive advantage throughout the transition from endosymbiosis to symbiogenesis. Based on the contribution of diatoms to ocean biogeochemical cycles, VHA-mediated enhancement of photosynthesis contributes at least 3.5 Gtons of fixed carbon per year (or 7% of primary production in the ocean), providing an example of a symbiosis-derived evolutionary innovation with global environmental implications.

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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.

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Previous studies have suggested that the capacity of natural dissolved organic carbon (DOC) molecules to interact with biological membranes is associated with their aromaticity (SAC340 ); origin (allochthonous versus autochthonous, FI); molecular weight (Abs254/365 ); and relative fluorescence of DOC moieties (PARAFAC analysis). These interactions may be especially important when fish are challenged by acidic waters, which are known to inhibit the active uptake of Na+ and Cl- , while stimulating diffusive ion losses in freshwater fishes. Therefore, zebrafish were acclimated (7 days, pH 7.0) to five natural DOC sources (10 mg C/L), two from the Amazon Basin and three from Canada, together with a "no-added DOC" control. After the acclimation, fish were challenged by exposure to acidic water (pH 4.0) for 3 h. Osmoregulatory parameters were measured at pH 7.0 and 4.0. Acclimation to the five DOC sources did not disturb Na+ , Cl- and ammonia net fluxes, but resulted in differential elevations in Na+ , K+ ATPase and v-type H+ ATPase activities in fish at pH 7.0. However, after transfer to pH.4.0, the control fish exhibited rapid increases in both enzymes. In contrast the DOC- acclimated animals exhibited unchanged (Na+ , K+ ATPase) or differentially increased (v-type H+ ATPase) activities. Na+ , Cl- and ammonia net fluxes remained unchanged in the control fish, but were differentially elevated in most of the DOC treatments at pH 4.0, relative to the same DOC treatments at pH 7.0. Correlations between the osmoregulatory data the DOCs properties highlight that the DOC properties drive different effects on gill physiology.

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In euryhaline teleosts, Na+, K+-ATPase (NKA) and V-type H + -ATPase A (VHA A) are important ion-transporters located in cell membrane. Lipid rafts (LR) are plasma membrane microdomains enriched in cholesterol, sphingolipids, and proteins (e.g., flotillin). Flotillin is a LR-associated protein, commonly used as the LR marker. Previous mammalian studies showed that LR may play a crucial role in ion exchanges. Meanwhile, studies on mammals and rainbow trout showed that NKA were found to be present mainly in LR. However, little is known about LR in fish. Therefore, the present study aimed to investigate the involvement of branchial LR in osmoregulation of tilapia and milkfish, two euryhaline teleosts with different salinity preferences, by (i) extracting LR from the gills of euryhaline teleosts; (ii) detecting the abundance of LR marker protein (flotillin-2) and ion-transporters (NKA and VHA A) in branchial LR and non-LR of fresh water- and seawater-acclimated milkfish and tilapia. The results indicated that the protein abundance of LR marker, flotillin-2, changed with environmental salinities in branchial LR of tilapia. In addition, flotillin-2 and NKA were only found in LR in both tilapia and milkfish gills, while VHA A were mainly present in non-LR. Relative protein abundance of NKA was found to be significantly higher in gills of freshwater milkfish and seawater tilapia, while VHA A was significantly higher in gills of freshwater tilapia and milkfish. This study illustrated differential distribution and salinity-dependent expression of NKA and VHA A in cell membrane of gill tissues of euryhaline teleosts with different salinity preferences.

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Cold acclimation increases cold tolerance of chill-susceptible insects and the acclimation response often involves improved organismal ion balance and osmoregulatory function at low temperature. However, the physiological mechanisms underlying plasticity of ion regulatory capacity are largely unresolved. Here we used Ussing chambers to explore the effects of cold exposure on hindgut KCl reabsorption in cold- (11 °C) and warm-acclimated (30 °C) Locusta migratoria. Cooling (from 30 to 10 °C) reduced active reabsorption across recta from warm-acclimated locusts, while recta from cold-acclimated locusts maintained reabsorption at 10 °C. The differences in transport capacity were not linked to major rearrangements of membrane phospholipid profiles. Yet, the stimulatory effect of two signal transduction pathways were altered by temperature and/or acclimation. cAMP-stimulation increased reabsorption in both acclimation groups, with a strong stimulatory effect at 30 °C and a moderate stimulatory effect at 10 °C. cGMP-stimulation also increased reabsorption in both acclimation groups at 30 °C, but their response to cGMP differed at 10 °C. Recta from warm-acclimated locusts, characterised by reduced reabsorption at 10 °C, recovered reabsorption capacity following cGMP-stimulation at 10 °C. In contrast, recta from cold-acclimated locusts, characterised by sustained reabsorption at 10 °C, were unaffected by cGMP-stimulation. Furthermore, cold-exposed recta from warm-acclimated locusts were insensitive to bafilomycin-α1, a V-type H+-ATPase inhibitor, whereas this blocker reduced reabsorption across cold-exposed recta from cold-acclimated animals. In conclusion, bafilomycin-sensitive and cGMP-dependent transport mechanism(s) are likely blocked during cold exposure in warm-acclimated animals while preserved in cold-acclimated animals. These may in part explain the large differences in rectal ion transport capacity between acclimation groups at low temperature.

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Formation water (FoW) is a by-product from oil and gas production and usually has high concentrations of soluble salts and metals. Calcium (Ca) and magnesium (Mg) have been shown to reduce the toxicity of metals to aquatic animals, and previous study showed that high waterborne Ca exerts mild effect against disturbances on Na+ regulation in Amazonian armored catfish tamoatá (Hoplosternum littorale) acutely exposed to high Fe, Mn, and Ba levels. Here, we hypothesized that high Mg levels might also reduce the toxic effects of these metals on Na+ regulation of tamoatá. The exposure to 5% FoW promoted an increase in Na+ uptake and a rapid accumulation of Na+ in all tissues analyzed (kidney

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This study investigated ionoregulatory strategies used by freshwater (FW) nymphs of the mayfly Hexagenia rigida Like other FW organisms, H. rigida nymphs maintain hemolymph ion levels (in mmol l-1: Na+ ∼102; Cl- ∼84; K+ ∼6; pH ∼7.35) far in excess of their surroundings. This appears to be accomplished by the combined actions of the alimentary canal, Malpighian tubules (MTs) and tracheal gills. The alimentary canal contributes in a region-specific manner, a view supported by: (1) spatial differences in the activity of basolateral Na+/K+-ATPase (NKA) and apical V-type H+-ATPase (VA) and (2) region-specific Na+ and K+ flux rates. Both indicate a prominent role for the hindgut (rectum) in K+ reabsorption. MTs also exhibit region-specific differences in Na+ and K+ flux rates that are coupled with an organized but tortuous architecture. NKA and VA activities were highest in MTs versus all other organs examined. Tracheal gills were found to be sites of Na+ uptake, but no difference in Na+ uptake was found between gills taken from different regions of the abdomen or spatially along individual gills. This is likely because each gill exhibited a dense population of NKA and/or VA immunoreactive cells (putative ionocytes). Data provide new insight into how FW mayfly nymphs regulate salt and water balance using the alimentary canal, MTs and tracheal gills as well as the first direct evidence that tracheal gills acquire ions from FW.

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Systemic acid-base balance and osmotic/ionic regulation in decapod crustaceans are in part maintained by a set of transport-related enzymes such as carbonic anhydrase (CA), Na+/K+-ATPase (NKA), H+-ATPase (HAT), Na+/K+/2Cl- cotransporter (NKCC), Na+/Cl-/HCO[Formula: see text] cotransporter (NBC), Na+/H+ exchanger (NHE), Arginine kinase (AK), Sarcoplasmic Ca+2-ATPase (SERCA) and Calreticulin (CRT). We carried out a comparative molecular analysis of these genes in three commercially important yet eco-physiologically distinct freshwater crayfish, Cherax quadricarinatus, C. destructor and C. cainii, with the aim to identify mutations in these genes and determine if observed patterns of mutations were consistent with the action of natural selection. We also conducted a tissue-specific expression analysis of these genes across seven different organs, including gills, hepatopancreas, heart, kidney, liver, nerve and testes using NGS transcriptome data. The molecular analysis of the candidate genes revealed a high level of sequence conservation across the three Cherax sp. Hyphy analysis revealed that all candidate genes showed patterns of molecular variation consistent with neutral evolution. The tissue-specific expression analysis showed that 46% of candidate genes were expressed in all tissue types examined, while approximately 10% of candidate genes were only expressed in a single tissue type. The largest number of genes was observed in nerve (84%) and gills (78%) and the lowest in testes (66%). The tissue-specific expression analysis also revealed that most of the master genes regulating pH and osmoregulation (CA, NKA, HAT, NKCC, NBC, NHE) were expressed in all tissue types indicating an important physiological role for these genes outside of osmoregulation in other tissue types. The high level of sequence conservation observed in the candidate genes may be explained by the important role of these genes as well as potentially having a number of other basic physiological functions in different tissue types.

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We analyzed V-type H+-ATPase (VA) and Na+/K+-ATPase (NKA) along the caeca and midgut of third instar Drosophila larvae using immunohistochemistry and ATPase activity assays. Corresponding H+ and K+ fluxes were characterized using the Scanning Ion-Selective Electrode Technique (SIET), and the roles of transport ATPases in energizing ion transport across the larval gut were investigated by basal application of bafilomycin, a VA inhibitor, and ouabain, a NKA inhibitor. Addition of bafilomycin led to a decrease in H+ absorption along the caeca and midgut except at the copper cells and large flat cell zone of the middle midgut. H+ absorption was decreased by acetazolamide, consistent with carbonic anhydrase activity in all regions except at the large flat cell zone of the middle midgut. Bafilomycin or acetazolamide also led to decreased K+ absorption across the caeca and the anterior midgut. Our data show the dependence of K+ transport on H+ gradients established by the VA in the latter regions, consistent with the presence of a Cation-Proton Antiporter (CPA2) identified in other insect epithelia. Addition of ouabain led to the increase of K+ absorption along the anterior midgut and the large flat cell zone of the middle midgut, suggesting a role for the NKA in these regions. This study shows the importance of both ATPases in driving ion transport across the gut of larval Drosophila.

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The excretion of nitrogenous waste products in the form of ammonia (NH3) and ammonium (NH4 (+)) is a fundamental process in aquatic organisms. For mytilid bivalves, little is known about the mechanisms and sites of excretion. This study investigated the localization and the mechanisms of ammonia excretion in mytilid mussels. An Rh protein was found to be abundantly expressed in the apical cell membrane of the plicate organ, which was previously described as a solely respiratory organ. The Rh protein was also expressed in the gill, although at significantly lower concentrations, but was not detectable in mussel kidney. Furthermore, NH3/NH4 (+) was not enriched in the urine, suggesting that kidneys are not involved in active NH3/NH4 (+) excretion. Exposure to elevated seawater pH of 8.5 transiently reduced NH3/NH4 (+) excretion rates, but they returned to control values following 24 h acclimation. These mussels had increased abundance of V-type H(+)-ATPase in the apical membranes of plicate organ cells; however, NH3/NH4 (+) excretion rates were not affected by the V-type H(+)-ATPase specific inhibitor concanamycin A (100 nmol l(-1)). In contrast, inhibition of ciliary beating with dopamine and increased seawater viscosity significantly reduced NH3 excretion rates under control pH (8.0). These results suggest that NH3/NH4 (+) excretion in mytilid mussels takes place by passive NH3 diffusion across respiratory epithelia via the Rh protein, facilitated by the water current produced for filter feeding, which prevents accumulation of NH3 in the boundary layer. This mechanism would be energy efficient for sessile organisms, as they already generate water currents for filter feeding.

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Humans possess three major isoforms of the apolipoprotein E (ApoE) gene encoded by three alleles: ApoE ɛ2 (ApoE2), ApoE ɛ3 (ApoE3), and ApoE ɛ4 (ApoE4). It is established that the three ApoE isoforms confer differential susceptibility to Alzheimer's disease (AD); however, an in-depth molecular understanding of the underlying mechanisms is currently unavailable. In this study, we examined the cortical proteome differences among the three ApoE isoforms using 6-month-old female, human ApoE2, ApoE3, and ApoE4 gene-targeted replacement mice and two-dimensional proteomic analyses. The results reveal that the three ApoE brains differ primarily in two areas: cellular bioenergetics and synaptic transmission. Of particular significance, we show for the first time that the three ApoE brains differentially express a key component of the catalytic domain of the V-type H+-ATPase (Atp6v), a proton pump that mediates the concentration of neurotransmitters into synaptic vesicles and thus is crucial in synaptic transmission. Specifically, our data demonstrate that ApoE2 brain exhibits significantly higher levels of the B subunit of Atp6v (Atp6v1B2) when compared to both ApoE3 and ApoE4 brains, with ApoE4 brain exhibiting the lowest expression. Our additional analyses show that Atp6v1B2 is significantly impacted by aging and AD pathology and the data suggest that Atp6v1B2 deficiency could be involved in the progressive loss of synaptic integrity during early development of AD. Collectively, our findings indicate that human ApoE isoforms differentially modulate regulatory mechanisms of bioenergetic and synaptic function in female brain. A more efficient and robust status in both areas-in which Atp6v may play a role-could serve as a potential mechanism contributing to the neuroprotective and cognition-favoring properties associated with the ApoE2 genotype.

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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.

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The transcripts of three putative ammonia (NH3/NH4 (+)) transporters, Rhesus-like glycoproteins AeRh50-1, AeRh50-2 and Amt/Mep-like AeAmt1 were detected in the anal papillae of larval Aedes aegypti Quantitative PCR studies revealed 12-fold higher transcript levels of AeAmt1 in anal papillae relative to AeRh50-1, and levels of AeRh50-2 were even lower. Immunoblotting revealed AeAmt1 in anal papillae as a pre-protein with putative monomeric and trimeric forms. AeAmt1 was immunolocalized to the basal side of the anal papillae epithelium where it co-localized with Na(+)/K(+)-ATPase. Ammonium concentration gradients were measured adjacent to anal papillae using the scanning ion-selective electrode technique (SIET) and used to calculate ammonia efflux by the anal papillae. dsRNA-mediated reductions in AeAmt1 decreased ammonia efflux at larval anal papillae and significantly increased ammonia levels in hemolymph, indicating a principal role for AeAmt1 in ammonia excretion. Pharmacological characterization of ammonia transport mechanisms in the anal papillae suggests that, in addition to AeAmt1, the ionomotive pumps V-type H(+)-ATPase and Na(+)/K(+)-ATPase as well as NHE3 are involved in ammonia excretion at the anal papillae.

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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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The evolutionary transition from water-breathing to air-breathing involved not only a change in function of the organs of respiratory gas exchange and N-waste excretion, but also in the organs of ion uptake from the environment. A combination of in vivo and in vitro techniques was used to look at the relative importance of the gills versus the gut in Na(+), Cl(-), and K(+) balance in two closely related erythrinid species: a facultative air-breather, the jeju (Hoplerythrinus unitaeniatus) and an obligate water-breather, the traira (Hoplias malabaricus). The jeju has a well-vascularized physostomous swimbladder, while that in the traira is poorly vascularized, but the gills are much larger. Both species are native to the Amazon and are common in the ion-poor, acidic blackwaters of the Rio Negro. Under fasting conditions, the traira was able to maintain positive net Na(+) and Cl(-) balance in this water, and only slightly negative net K(+) balance. However, the jeju was in negative net balance for all three ions and had lower plasma Na(+) and Cl(-) concentrations, despite exhibiting higher branchial Na(+), K(+)ATPase and v-type H(+)ATPase activities. In the intestine, activities of these same enzymes were also higher in the jeju, and in vitro measurements of net area-specific rates of Na(+), Cl(-), and K(+) absorption, as well as the overall intestinal absorption capacities for these three ions, were far greater than in the traira. When acutely exposed to disturbances in water O2 levels (severe hypoxia ~15% or hyperoxia ~420% saturation), gill ionoregulation was greatly perturbed in the traira but less affected in the jeju, which could "escape" the stressor by voluntarily air-breathing. We suggest that a shift of ionoregulatory capacity from the gills to the gut may have occurred in the evolutionary transition to air-breathing in jeju, and in consequence branchial ionoregulation, while less powerful, is also less impacted by variations in water O2 levels.

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Hydrothermal vent organisms have evolved physiological adaptations to cope with extreme abiotic conditions including temperature and pH. To date, acid-base regulatory abilities of vent organisms are poorly investigated, although this physiological feature is essential for survival in low pH environments. We report the acid-base regulatory mechanisms of a hydrothermal vent crab, Xenograpsus testudinatus, endemic to highly acidic shallow-water vent habitats with average environment pH-values ranging between 5.4 and 6.6. Within a few hours, X. testudinatus restores extracellular pH (pHe) in response to environmental acidification of pH 6.5 (1.78 kPa pCO2) accompanied by an increase in blood [Formula: see text] levels from 8.8 ± 0.3 to 31 ± 6 mM. Branchial Na(+)/K(+)-ATPase (NKA) and V-type H(+)-ATPase (VHA), the major ion pumps involved in branchial acid-base regulation, showed dynamic increases in response to acidified conditions on the mRNA, protein and activity level. Immunohistochemical analyses demonstrate the presence of NKA in basolateral membranes, whereas the VHA is predominantly localized in cytoplasmic vesicles of branchial epithelial- and pillar-cells. X. testudinatus is closely related to other strong osmo-regulating brachyurans, which is also reflected in the phylogeny of the NKA. Accordingly, our results suggest that the evolution of strong ion regulatory abilities in brachyuran crabs that allowed the occupation of ecological niches in euryhaline, freshwater, and terrestrial habitats are probably also linked to substantial acid-base regulatory abilities. This physiological trait allowed X. testudinatus to successfully inhabit one of the world's most acidic marine environments.

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Symbiotic dinoflagellate algae residing inside coral tissues supply the host with the majority of their energy requirements through the translocation of photosynthetically fixed carbon. The algae, in turn, rely on the host for the supply of inorganic carbon. Carbon must be concentrated as CO2 in order for photosynthesis to proceed, and here we show that the coral host plays an active role in this process. The host-derived symbiosome membrane surrounding the algae abundantly expresses vacuolar H(+)-ATPase (VHA), which acidifies the symbiosome space down to pH ∼ 4. Inhibition of VHA results in a significant decrease in average H(+) activity in the symbiosome of up to 75% and a significant reduction in O2 production rate, a measure of photosynthetic activity. These results suggest that host VHA is part of a previously unidentified carbon concentrating mechanism for algal photosynthesis and provide mechanistic evidence that coral host cells can actively modulate the physiology of their symbionts.

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Studies of euryhaline crustaceans have identified conserved osmoregulatory adaptions allowing hyper-osmoregulation in dilute waters. However, previous studies have mainly examined decapod brachyurans with marine ancestries inhabiting estuaries or tidal creeks on a seasonal basis. Here, we describe osmoregulation in the atyid Halocaridina rubra, an endemic Hawaiian shrimp of freshwater ancestry from the islands' anchialine ecosystem (coastal ponds with subsurface freshwater and seawater connections) that encounters near-continuous spatial and temporal salinity changes. Given this, survival and osmoregulatory responses were examined over a wide salinity range. In the laboratory, H. rubra tolerated salinities of ~0-56‰, acting as both a hyper- and hypo-osmoregulator and maintaining a maximum osmotic gradient of ~868 mOsm kg(-1) H2O in freshwater. Furthermore, hemolymph osmolality was more stable during salinity transfers relative to other crustaceans. Silver nitrate and vital mitochondria-rich cell staining suggest all gills are osmoregulatory, with a large proportion of each individual gill functioning in ion transport (including when H. rubra acts as an osmoconformer in seawater). Additionally, expression of ion transporters and supporting enzymes that typically undergo upregulation during salinity transfer in osmoregulatory gills (i.e. Na(+)/K(+)-ATPase, carbonic anhydrase, Na(+)/K(+)/2Cl(-) cotransporter, V-type H(+)-ATPase and arginine kinase) were generally unaltered in H. rubra during similar transfers. These results suggest H. rubra (and possibly other anchialine species) maintains high, constitutive levels of gene expression and ion transport capability in the gills as a means of potentially coping with the fluctuating salinities that are encountered in anchialine habitats. Thus, anchialine taxa represent an interesting avenue for future physiological research.

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