The silent loss of cell physiology hampers marine biosciences.

An ongoing loss of experts in marine cellular biochemistry and physiology (CBP) is stagnating the generation of knowledge upon which rapidly growing "omics" approaches rely, ultimately hampering our ability to predict organismal responses to climate change.

Gill ionocyte remodeling mediates blood pH regulation in rockfish (Sebastes diploproa) exposed to environmentally relevant hypercapnia.

Marine fishes excrete excess H+ using basolateral Na+-K+-ATPase (NKA) and apical Na+/H+ exchanger 3 (NHE3) in gill ionocytes. However, the mechanisms that regulate H+ excretion during exposure to environmentally relevant hypercapnia (ERH) remain poorly understood. Here, we explored transcriptomic, proteomic, and cellular responses in gills of juvenile splitnose rockfish (Sebastes diploproa) exposed to 3 days of ERH conditions (pH ∼7.5, ∼1,600 µatm Pco2). Blood pH was fully regulated at ∼7.75 despite a lack of significant changes in gill 1) mRNAs coding for proteins involved in blood acid-base regulation, 2) total NKA and NHE3 protein abundance, and 3) ionocyte density. However, ERH-exposed rockfish demonstrated increased NKA and NHE3 abundance on the ionocyte plasma membrane coupled with wider apical membranes and greater extension of apical microvilli. The observed gill ionocyte remodeling is consistent with enhanced H+ excretion that maintains blood pH homeostasis during exposure to ERH and does not necessitate changes at the expression or translation levels. These mechanisms of phenotypic plasticity may allow fishes to regulate blood pH during environmentally relevant acid-base challenges and thus have important implications for both understanding how organisms respond to climate change and for selecting appropriate metrics to evaluate its impact on marine ecosystems.NEW & NOTEWORTHY Splitnose rockfish exposed to environmentally relevant hypercapnia utilize existing proteins (rather than generate additional machinery) to maintain homeostasis.

Immunohistochemical and ultrastructural characterization of the inner ear epithelial cells of splitnose rockfish (Sebastes diploproa).

The inner ear of teleost fish regulates the ionic and acid-base chemistry and secretes protein matrix into the endolymph to facilitate otolith biomineralization, which is used to maintain vestibular and auditory functions. The otolith is biomineralized in a concentric ring pattern corresponding to seasonal growth, and this calcium carbonate (CaCO3) polycrystal has become a vital aging and life-history tool for fishery managers, ecologists, and conservation biologists. Moreover, biomineralization patterns are sensitive to environmental variability including climate change, thereby threatening the accuracy and relevance of otolith-reliant toolkits. However, the cellular biology of the inner ear is poorly characterized, which is a hurdle for a mechanistic understanding of the underlying processes. This study provides a systematic characterization of the cell types in the inner ear of splitnose rockfish (Sebastes diploproa). Scanning electron microscopy revealed the apical morphologies of six inner ear cell types. In addition, immunostaining and confocal microscopy characterized the expression and subcellular localization of the proteins Na+-K+-ATPase, carbonic anhydrase, V-type H+-ATPase, Na+-K+-2Cl--cotransporter, otolith matrix protein 1, and otolin-1 in six inner ear cell types bordering the endolymph. This fundamental cytological characterization of the rockfish inner ear epithelium illustrates the intricate physiological processes involved in otolith biomineralization and highlights how greater mechanistic understanding is necessary to predict their multistressor responses to future climate change.

Soluble adenylyl cyclase coordinates intracellular pH homeostasis and biomineralization in calcifying cells of a marine animal.

Biomineralizing cells concentrate dissolved inorganic carbon (DIC) and remove protons from the site of mineral precipitation. However, the molecular regulatory mechanisms that orchestrate pH homeostasis and biomineralization of calcifying cells are poorly understood. Here, we report that the acid-base sensing enzyme soluble adenylyl cyclase (sAC) coordinates intracellular pH (pHi) regulation in the calcifying primary mesenchyme cells (PMCs) of sea urchin larvae. Single-cell transcriptomics, in situ hybridization, and immunocytochemistry elucidated the spatiotemporal expression of sAC during skeletogenesis. Live pHi imaging of PMCs revealed that the downregulation of sAC activity with two structurally unrelated small molecules inhibited pHi regulation of PMCs, an effect that was rescued by the addition of cell-permeable cAMP. Pharmacological sAC inhibition also significantly reduced normal spicule growth and spicule regeneration, establishing a link between PMC pHi regulation and biomineralization. Finally, increased expression of sAC mRNA was detected during skeleton remineralization and exposure to CO2-induced acidification. These findings suggest that transcriptional regulation of sAC is required to promote remineralization and to compensate for acidic stress. This work highlights the central role of sAC in coordinating acid-base regulation and biomineralization in calcifying cells of a marine animal.

A novel perspective on the evolutionary loss of plasma-accessible carbonic anhydrase at the teleost gill.

The gills of most teleost fishes lack plasma-accessible carbonic anhydrase (paCA) that could participate in CO2 excretion. We tested the prevailing hypothesis that paCA would interfere with red blood cell (RBC) intracellular pH regulation by ß-adrenergic sodium-proton exchangers (ß-NHE) that protect pH-sensitive haemoglobin-oxygen (Hb-O2) binding during an acidosis. In an open system that mimics the gills, ß-NHE activity increased Hb-O2 saturation during a respiratory acidosis in the presence or absence of paCA, whereas the effect was abolished by NHE inhibition. However, in a closed system that mimics the tissue capillaries, paCA disrupted the protective effects of ß-NHE activity on Hb-O2 binding. The gills are an open system, where CO2 generated by paCA can diffuse out and is not available to acidifying the RBCs. Therefore, branchial paCA in teleosts may not interfere with RBC pH regulation by ß-NHEs, and other explanations for the evolutionary loss of the enzyme must be considered.

Evolving views of ionic, osmotic and acid-base regulation in aquatic animals.

The regulation of ionic, osmotic and acid-base (IOAB) conditions in biological fluids is among the most fundamental functions in all organisms; being surrounded by water uniquely shapes the IOAB regulatory strategies of water-breathing animals. Throughout its centennial history, Journal of Experimental Biology has established itself as a premier venue for publication of comparative, environmental and evolutionary studies on IOAB regulation. This Review provides a synopsis of IOAB regulation in aquatic animals, some of the most significant research milestones in the field, and evolving views about the underlying cellular mechanisms and their evolutionary implications. It also identifies promising areas for future research and proposes ideas for enhancing the impact of aquatic IOAB research.

Rapid blood acid-base regulation by European sea bass (Dicentrarchus labrax) in response to sudden exposure to high environmental CO2.

Fish in coastal ecosystems can be exposed to acute variations in CO2 of between 0.2 and 1 kPa CO2 (2000-10,000 µatm). Coping with this environmental challenge will depend on the ability to rapidly compensate for the internal acid-base disturbance caused by sudden exposure to high environmental CO2 (blood and tissue acidosis); however, studies about the speed of acid-base regulatory responses in marine fish are scarce. We observed that upon sudden exposure to ∼1 kPa CO2, European sea bass (Dicentrarchus labrax) completely regulate erythrocyte intracellular pH within ∼40 min, thus restoring haemoglobin-O2 affinity to pre-exposure levels. Moreover, blood pH returned to normal levels within ∼2 h, which is one of the fastest acid-base recoveries documented in any fish. This was achieved via a large upregulation of net acid excretion and accumulation of HCO3- in blood, which increased from ∼4 to ∼22 mmol l-1. While the abundance and intracellular localisation of gill Na+/K+-ATPase (NKA) and Na+/H+ exchanger 3 (NHE3) remained unchanged, the apical surface area of acid-excreting gill ionocytes doubled. This constitutes a novel mechanism for rapidly increasing acid excretion during sudden blood acidosis. Rapid acid-base regulation was completely prevented when the same high CO2 exposure occurred in seawater with experimentally reduced HCO3- and pH, probably because reduced environmental pH inhibited gill H+ excretion via NHE3. The rapid and robust acid-base regulatory responses identified will enable European sea bass to maintain physiological performance during large and sudden CO2 fluctuations that naturally occur in coastal environments.

Adrenergically induced translocation of red blood cell ß-adrenergic sodium-proton exchangers has ecological relevance for hypoxic and hypercapnic white seabass.

White seabass (Atractoscion nobilis) increasingly experience periods of low oxygen (O2; hypoxia) and high carbon dioxide (CO2, hypercapnia) due to climate change and eutrophication of the coastal waters of California. Hemoglobin (Hb) is the principal O2 carrier in the blood and in many teleost fishes Hb-O2 binding is compromised at low pH; however, the red blood cells (RBC) of some species regulate intracellular pH with adrenergically stimulated sodium-proton-exchangers (ß-NHEs). We hypothesized that RBC ß-NHEs in white seabass are an important mechanism that can protect the blood O2-carrying capacity during hypoxia and hypercapnia. We determined the O2-binding characteristics of white seabass blood, the cellular and subcellular response of RBCs to adrenergic stimulation, and quantified the protective effect of ß-NHE activity on Hb-O2 saturation. White seabass had typical teleost Hb characteristics, with a moderate O2 affinity (Po2 at half-saturation; P50 2.9 kPa) that was highly pH-sensitive (Bohr coefficient -0.92; Root effect 52%). Novel findings from super-resolution microscopy revealed ß-NHE protein in vesicle-like structures and its translocation into the membrane after adrenergic stimulation. Microscopy data were corroborated by molecular and phylogenetic results and a functional characterization of ß-NHE activity. The activation of RBC ß-NHEs increased Hb-O2 saturation by ∼8% in normoxic hypercapnia and by up to ∼20% in hypoxic normocapnia. Our results provide novel insight into the cellular mechanism of adrenergic RBC stimulation within an ecologically relevant context. ß-NHE activity in white seabass has great potential to protect arterial O2 transport during hypoxia and hypercapnia but is less effective during combinations of these stressors.

Regulation of coral calcification by the acid-base sensing enzyme soluble adenylyl cyclase.

Coral calcification is intricately linked to the chemical composition of the fluid in the extracellular calcifying medium (ECM), which is situated between the calcifying cells and the skeleton. Here we demonstrate that the acid-base sensing enzyme soluble adenylyl cyclase (sAC) is expressed in calcifying cells of the coral Stylophora pistillata. Furthermore, pharmacological inhibition of sAC in coral microcolonies resulted in acidification of the ECM as estimated by the pH-sensitive ratiometric indicator SNARF, and decreased calcification rates, as estimated by calcein labeling of crystal growth. These results indicate that sAC activity modulates some of the molecular machinery involved in producing the coral skeleton, which could include ion-transporting proteins and vesicular transport. To our knowledge this is the first study to directly demonstrate biological regulation of the alkaline pH of the coral ECM and its correlation with calcification.

Dynamic subcellular translocation of V-type H+ -ATPase is essential for biomineralization of the diatom silica cell wall.

Diatom cell walls, called frustules, are main sources of biogenic silica in the ocean and their intricate morphology is an inspiration for nanoengineering. Here we show dynamic aspects of frustule biosynthesis involving acidification of the silica deposition vesicle (SDV) by V-type H+  ATPase (VHA). Transgenic Thalassiosira pseudonana expressing the VHA B subunit tagged with enhanced green fluorescent protein (VHAB -eGFP) enabled subcellular protein localization in live cells. In exponentially growing cultures, VHAB -eGFP was present in various subcellular localizations including the cytoplasm, SDVs and vacuoles. We studied the role of VHA during frustule biosynthesis in synchronized cell cultures of T. pseudonana. During the making of new biosilica components, VHAB -eGFP first localized in the girdle band SDVs, and subsequently in valve SDVs. In single cell time-lapse imaging experiments, VHAB -eGFP localization in SDVs precluded accumulation of the acidotropic silica biomineralization marker PDMPO. Furthermore, pharmacological VHA inhibition prevented PDMPO accumulation in the SDV, frustule biosynthesis and cell division, as well as insertion of the silicalemma-associated protein SAP1 into the SDVs. Finally, partial inhibition of VHA activity affected the nanoscale morphology of the valve. Altogether, these results indicate that VHA is essential for frustule biosynthesis by acidifying the SDVs and regulating the insertion of other structural proteins into the SDV.

Differential glycogen utilization in shark acid- and base-regulatory gill cells.

Na+/K+-ATPase (NKA)- and vacuolar H+-ATPase (VHA)-rich cells in shark gills secrete excess acid and base, respectively, to seawater to maintain blood acid-base homeostasis. Both cell types are rich in mitochondria, indicating high ATP demand; however, their metabolic fuel is unknown. Here, we report that NKA- and VHA-rich cells have large glycogen stores. Glycogen abundance in NKA-rich cells was lower in starved sharks compared with 24 h post-fed sharks, reflecting higher energy demand for acid secretion during normal activity and glycogen replenishment during the post-feeding period. Conversely, glycogen abundance in VHA-rich cells was high in starved sharks and it became depleted post-feeding. Furthermore, inactive cells with cytoplasmic VHA had large glycogen stores and active cells with basolateral VHA had depleted glycogen stores. These results indicate that glycogen is a main energy source in both NKA- and VHA-rich cells, and point to differential energy use associated with net acid and net base secretion, respectively.

Regulation of glyceraldehyde-3-phosphate dehydrogenase by hypoxia inducible factor 1 in the white shrimp Litopenaeus vannamei during hypoxia and reoxygenation.

Hypoxia is a frequent source of stress in the estuarine habitat of the white shrimp Litopenaeus vannamei. During hypoxia, L. vannamei gill cells rely more heavily on anaerobic glycolysis to obtain ATP. This is mediated by transcriptional up-regulation of glycolytic enzymes including glyceraldehyde-3-phosphate dehydrogenase (GAPDH). The hypoxia inducible factor 1 (HIF-1) is an important transcriptional activator of several glycolytic enzymes during hypoxia in diverse animals, including crustaceans. In this work, we cloned and sequenced a fragment corresponding to the 5' flank of the GAPDH gene and identified a putative HIF-1 binding site, as well as sites for other transcription factors involved in the hypoxia signaling pathway. To investigate the role of HIF-1 in GAPDH regulation, we simultaneously injected double-stranded RNA (dsRNA) into shrimp to silence HIF-1α and HIF-1ß under normoxia, hypoxia, and hypoxia followed by reoxygenation, and then measured gill HIF-1α, HIF-1ß expression, GAPDH expression and activity, and glucose and lactate concentrations at 0, 3, 24 and 48 h. During normoxia, HIF-1 silencing induced up-regulation of GAPDH transcripts and activity, suggesting that expression is down-regulated via HIF-1 under these conditions. In contrast, HIF-1 silencing during hypoxia abolished the increases in GAPDH expression and activity, glucose and lactate concentrations. Finally, HIF-1 silencing during hypoxia-reoxygenation prevented the increase in GAPDH expression, however, those changes were not reflected in GAPDH activity and lactate accumulation. Altogether, these results indicate that GAPDH and glycolysis are transcriptionally regulated by HIF-1 in gills of white shrimp.

Symbiont photosynthesis in giant clams is promoted by V-type H+-ATPase from host cells.

Giant clams (genus Tridacna) are the largest living bivalves and, like reef-building corals, host symbiotic dinoflagellate algae (Symbiodinium) that significantly contribute to their energy budget. In turn, Symbiodinium rely on the host to supply inorganic carbon (Ci) for photosynthesis. In corals, host 'proton pump' vacuolar-type H+-ATPase (VHA) is part of a carbon-concentrating mechanism (CCM) that promotes Symbiodinium photosynthesis. Here, we report that VHA in the small giant clam (Tridacna maxima) similarly promotes Symbiodinium photosynthesis. VHA was abundantly expressed in the apical membrane of epithelial cells of T. maxima's siphonal mantle tubule system, which harbors Symbiodinium Furthermore, application of the highly specific pharmacological VHA inhibitors bafilomycin A1 and concanamycin A significantly reduced photosynthetic O2 production by ∼40%. Together with our observation that exposure to light increased holobiont aerobic metabolism ∼5-fold, and earlier estimates that translocated fixed carbon exceeds metabolic demand, we conclude that VHA activity in the siphonal mantle confers strong energetic benefits to the host clam through increased supply of Ci to algal symbionts and subsequent photosynthetic activity. The convergent role of VHA in promoting Symbiodinium photosynthesis in the giant clam siphonal mantle tubule system and coral symbiosome suggests that VHA-driven CCM is a common exaptation in marine photosymbioses that deserves further investigation in other taxa.

Acid secretion by the boring organ of the burrowing giant clam, Tridacna crocea.

The giant clam Tridacna crocea, native to Indo-Pacific coral reefs, is noted for its unique ability to bore fully into coral rock and is a major agent of reef bioerosion. However, T. crocea's mechanism of boring has remained a mystery despite decades of research. By exploiting a new, two-dimensional pH-sensing technology and manipulating clams to press their presumptive boring tissue (the pedal mantle) against pH-sensing foils, we show that this tissue lowers the pH of surfaces it contacts by greater than or equal to 2 pH units below seawater pH day and night. Acid secretion is likely mediated by vacuolar-type H+-ATPase, which we demonstrate (by immunofluorescence) is abundant in the pedal mantle outer epithelium. Our discovery of acid secretion solves this decades-old mystery and reveals that, during bioerosion, T. crocea can liberate reef constituents directly to the soluble phase, rather than producing sediment alone as earlier assumed.

Coral host cells acidify symbiotic algal microenvironment to promote photosynthesis.

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.

Identification of a molecular pH sensor in coral.

Maintaining stable intracellular pH (pHi) is essential for homeostasis, and requires the ability to both sense pH changes that may result from internal and external sources, and to regulate downstream compensatory pH pathways. Here we identified the cAMP-producing enzyme soluble adenylyl cyclase (sAC) as the first molecular pH sensor in corals. sAC protein was detected throughout coral tissues, including those involved in symbiosis and calcification. Application of a sAC-specific inhibitor caused significant and reversible pHi acidosis in isolated coral cells under both dark and light conditions, indicating sAC is essential for sensing and regulating pHi perturbations caused by respiration and photosynthesis. Furthermore, pHi regulation during external acidification was also dependent on sAC activity. Thus, sAC is a sensor and regulator of pH disturbances from both metabolic and external origin in corals. Since sAC is present in all coral cell types, and the cAMP pathway can regulate virtually every aspect of cell physiology through post-translational modifications of proteins, sAC is likely to trigger multiple homeostatic mechanisms in response to pH disturbances. This is also the first evidence that sAC modulates pHi in any non-mammalian animal. Since corals are basal metazoans, our results indicate this function is evolutionarily conserved across animals.

Acid-base physiology, neurobiology and behaviour in relation to CO2-induced ocean acidification.

Experimental exposure to ocean and freshwater acidification affects the behaviour of multiple aquatic organisms in laboratory tests. One proposed cause involves an imbalance in plasma chloride and bicarbonate ion concentrations as a result of acid-base regulation, causing the reversal of ionic fluxes through GABAA receptors, which leads to altered neuronal function. This model is exclusively based on differential effects of the GABAA receptor antagonist gabazine on control animals and those exposed to elevated CO2 However, direct measurements of actual chloride and bicarbonate concentrations in neurons and their extracellular fluids and of GABAA receptor properties in aquatic organisms are largely lacking. Similarly, very little is known about potential compensatory mechanisms, and about alternative mechanisms that might lead to ocean acidification-induced behavioural changes. This article reviews the current knowledge on acid-base physiology, neurobiology, pharmacology and behaviour in relation to marine CO2-induced acidification, and identifies important topics for future research that will help us to understand the potential effects of predicted levels of aquatic acidification on organisms.

Dopamine D1 receptor activation leads to object recognition memory in a coral reef fish.

Object recognition memory is the ability to identify previously seen objects and is an adaptive mechanism that increases survival for many species throughout the animal kingdom. Previously believed to be possessed by only the highest order mammals, it is now becoming clear that fish are also capable of this type of memory formation. Similar to the mammalian hippocampus, the dorsolateral pallium regulates distinct memory processes and is modulated by neurotransmitters such as dopamine. Caribbean bicolour damselfish (Stegastes partitus) live in complex environments dominated by coral reef structures and thus likely possess many types of complex memory abilities including object recognition. This study used a novel object recognition test in which fish were first presented two identical objects, then after a retention interval of 10 min with no objects, the fish were presented with a novel object and one of the objects they had previously encountered in the first trial. We demonstrate that the dopamine D1-receptor agonist (SKF 38393) induces the formation of object recognition memories in these fish. Thus, our results suggest that dopamine-receptor mediated enhancement of spatial memory formation in fish represents an evolutionarily conserved mechanism in vertebrates.

Soluble adenylyl cyclase is an acid-base sensor in epithelial base-secreting cells.

Blood acid-base regulation by specialized epithelia, such as gills and kidney, requires the ability to sense blood acid-base status. Here, we developed primary cultures of ray (Urolophus halleri) gill cells to study mechanisms for acid-base sensing without the interference of whole animal hormonal regulation. Ray gills have abundant base-secreting cells, identified by their noticeable expression of vacuolar-type H(+)-ATPase (VHA), and also express the evolutionarily conserved acid-base sensor soluble adenylyl cyclase (sAC). Exposure of cultured cells to extracellular alkalosis (pH 8.0, 40 mM HCO3 (-)) triggered VHA translocation to the cell membrane, similar to previous reports in live animals experiencing blood alkalosis. VHA translocation was dependent on sAC, as it was blocked by the sAC-specific inhibitor KH7. Ray gill base-secreting cells also express transmembrane adenylyl cyclases (tmACs); however, tmAC inhibition by 2',5'-dideoxyadenosine did not prevent alkalosis-dependent VHA translocation, and tmAC activation by forskolin reduced the abundance of VHA at the cell membrane. This study demonstrates that sAC is a necessary and sufficient sensor of extracellular alkalosis in ray gill base-secreting cells. In addition, this study indicates that different sources of cAMP differentially modulate cell biology.

Novel and potential physiological roles of vacuolar-type H+-ATPase in marine organisms.

The vacuolar-type H(+)-ATPase (VHA) is a multi-subunit enzyme that uses the energy from ATP hydrolysis to transport H(+) across biological membranes. VHA plays a universal role in essential cellular functions, such as the acidification of lysosomes and endosomes. In addition, the VHA-generated H(+)-motive force can drive the transport of diverse molecules across cell membranes and epithelia for specialized physiological functions. Here, I discuss diverse physiological functions of VHA in marine animals, focusing on recent discoveries about base secretion in shark gills, potential bone dissolution by Osedax bone-eating worms and its participation in a carbon-concentrating mechanism that promotes coral photosynthesis. Because VHA is evolutionarily conserved among eukaryotes, it is likely to play many other essential physiological roles in diverse marine organisms. Elucidating and characterizing basic VHA-dependent mechanisms could help to determine species responses to environmental stress, including (but not limited to) that resulting from climate change.