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Luquet, CM, Genovese G, Tresguerres M.  In Press.  Gas exchange and acid-base balance. Neohelice granulata A model species for biological studies on crustaceans. ( Rodriguez E, Luppi T, Eds.)., UK: Cambridge Scholars Publishing
Kwan, GT, Finnerty S, Wegner NC, Tresguerres M.  2019.  Quantification of cutaneous ionocytes in small aquatic organisms. bio-protocol. 9(9):e3227.   10.21769/BioProtoc.3227   Abstract

Aquatic organisms have specialized cells called ionocytes that regulate the ionic composition, osmolarity, and acid/base status of internal fluids. In small aquatic organisms such as fishes in their early life stages, ionocytes are typically found on the cutaneous surface and their abundance can change to help cope with various metabolic and environmental factors. Ionocytes profusely express ATPase enzymes, most notably Na+/K+ ATPase, which can be identified by immunohistochemistry. However, quantification of cutaneous ionocytes is not trivial due to the limited camera’s focal plane and the microscope’s field-of-view. This protocol describes a technique to consistently and reliably identify, image, and measure the relative surface area covered by cutaneous ionocytes through software-mediated focus-stacking and photo-stitching–thereby allowing the quantification of cutaneous ionocyte area as a proxy for ion transporting capacity across the skin. Because ionocytes are essential for regulating ionic composition, osmolarity, and acid/base status of internal fluids, this technique is useful for studying physiological mechanisms used by fish larvae and other small aquatic organisms during development and in response to environmental stress.

Kwan, GT, Wexler JB, Wegner NC, Tresguerres M.  2019.  Ontogenetic changes in cutaneous and branchial ionocytes and morphology in yellowfin tuna (Thunnus albacares) larvae. J Comp Physiol B. 189(1):81-95.   AbstractWebsite

The development of osmoregulatory and gas exchange organs was studied in larval yellowfin tuna (Thunnus albacares) from 2 to 25 days post-hatching (2.9–24.5 mm standard length, SL). Cutaneous and branchial ionocytes were identified using Na+/K+-ATPase immunostaining and scanning electron microscopy. Cutaneous ionocyte abundance significantly increased with SL, but a reduction in ionocyte size and density resulted in a significant decrease in relative ionocyte area. Cutaneous ionocytes in preflexion larvae had a wide apical opening with extended microvilli; however, microvilli retracted into an apical pit from flexion onward. Lamellae in the gill and pseudobranch were first detected ~ 3.3 mm SL. Ionocytes were always present on the gill arch, first appeared in the filaments and lamellae of the pseudobranch at 3.4 mm SL, and later in gill filaments at 4.2 mm SL, but were never observed in the gill lamellae. Unlike the cutaneous ionocytes, gill and pseudobranch ionocytes had a wide apical opening with extended microvilli throughout larval development. The interlamellar fusion, a specialized gill structure binding the lamellae of ram-ventilating fish, began forming by ~ 24.5 mm SL and contained ionocytes, a localization never before reported. Ionocytes were retained on the lamellar fusions and also found on the filament fusions of larger sub-adult yellowfin tuna; however, sub-adult gill ionocytes had apical pits. These results indicate a shift in gas exchange and NaCl secretion from the skin to branchial organs around the flexion stage, and reveal novel aspects of ionocyte localization and morphology in ram-ventilating fishes.

Roa, JN, Tresguerres M.  2019.  Differential glycogen utilization in shark acid- and base-regulatory gill cells. J. Exp. Biol. 222:jeb199448.   AbstractWebsite

Na+/K+-ATPase (NKA)- and vacuolar H+-ATPase (VHA)-rich cells in shark gills respectively secrete excess acid and base 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 to 24h 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 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.

Camacho-Jiménez, L, Layva-Carrillo L, Peregrino-Uriarte AB, Duarte-Gutiérrez JL, Tresguerres M, Yepiz-Plascencia G.  2019.  Regulation of glyceraldehyde-3-phosphate dehydrogenase by hypoxia inducible factor 1 in the white shrimp Litopenaeus vannamei during hypoxia and reoxygenation. Comp. Biochem. Physiol. A. 235:56-65.   AbstractWebsite

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-1β under normoxia, hypoxia, and hypoxia followed by reoxygenation, and then measured gill HIF-1α, HIF-1β expression, and 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.

Barron, ME, Thies AB, Espinoza JA, Barott KL, Hamdoun A, Tresguerres M.  2018.  A vesicular Na+/Ca2+ exchanger in coral calcifying cells. PLoS One. 13(10):e0205367.   Abstract

The calcium carbonate skeletons of corals provide the underlying structure of coral reefs; however, the cellular mechanisms responsible for coral calcification remain poorly understood. In osteoblasts from vertebrate animals, a Na+/Ca2+ exchanger (NCX) present in the plasma membrane transports Ca2+ to the site of bone formation. The aims of this study were to establish whether NCX exists in corals and its localization within coral cells, which are essential first steps to investigate its potential involvement in calcification. Data mining identified genes encoding for NCX proteins in multiple coral species, a subset of which were more closely related to NCXs from vertebrates (NCXA). We cloned NCXA from Acropora yongei (AyNCXA), which, unexpectedly, contained a peptide signal that targets proteins to vesicles from the secretory pathway. AyNCXA subcellular localization was confirmed by heterologous expression of fluorescently tagged AyNCXA protein in sea urchin embryos, which localized together with known markers of intracellular vesicles. Finally, immunolabeling of coral tissues with specific antibodies revealed AyNCXA was present throughout coral tissue. AyNCXA was especially abundant in calcifying cells, where it exhibited a subcellular localization pattern consistent with intracellular vesicles. Altogether, our results demonstrate AyNCXA is present in vesicles in coral calcifying cells, where potential functions include intracellular Ca2+ homeostasis and Ca2+ transport to the growing skeleton as part of an intracellular calcification mechanism.

Hill, RW, Armstrong EJ, Inaba K, Morita M, Tresguerres M, Stillman JH, Roa JN, Kwan GT.  2018.  Acid secretion by the boring organ of the burrowing giant clam, Tridacna crocea. Biology Letters. 14:20180047.   10.1098/rsbl.2018.0047   AbstractWebsite

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 Hþ-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.

Armstrong, EJ, Roa JN, Stillman JH, Tresguerres M.  2018.  Symbiont photosynthesis in giant clams is promoted by V-type H+-ATPase from host cells. J. Exp. Biol.. 221:jeb177220.   doi:10.1242/jeb.177220   Abstract

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.

Tresguerres, M, Salmeron C.  2018.  Molecular, enzymatic, and cellular characterization of soluble adenylyl cyclase from aquatic animals. Methods in Enzymology . 605:525-549.   10.1016/bs.mie.2018.02.022   Abstract

The enzyme soluble adenylyl cyclase (sAC) is the most recently identified source of the messenger molecule cyclic adenosine monophosphate. sAC is evolutionarily conserved from cyanobacteria to human, is directly stimulated by [Formula: see text] ions, and can act as a sensor of environmental and metabolic CO2, pH, and [Formula: see text] levels. sAC genes tend to have multiple alternative promoters, undergo extensive alternative splicing, be translated into low mRNA levels, and the numerous sAC protein isoforms may be present in various subcellular localizations. In aquatic organisms, sAC has been shown to mediate various functions including intracellular pH regulation in coral, blood acid/base regulation in shark, heart beat rate in hagfish, and NaCl absorption in fish intestine. Furthermore, sAC is present in multiple other species and tissues, and sAC protein and enzymatic activity have been reported in the cytoplasm, the nucleus, and other subcellular compartments, suggesting even more diverse physiological roles. Although the methods and experimental tools used to study sAC are conventional, the complexity of sAC genes and proteins requires special considerations that are discussed in this chapter.

Hamilton, TJ, Kline DI, Tresguerres M.  2018.  Shoaling behaviour is differentially altered by ethanol and dopamine D1 receptor modulators in tropical marine forage fish. Canadian Journal of Fisheries and Aquatic Sciences. 56(3):1-6.   AbstractWebsite

Anchovies are filter-feeding fish that inhabit nearshore environments worldwide. With increasing human pharmaceutical use, drugs that alter neurological functioning are becoming more prevalent in aquatic ecosystems via wastewater effluent, creating the need for tests that can reliably determine sublethal effects of these drugs on coastal fish populations. In this study, we used Caribbean anchovies (Anchoa spp.) as a tropical marine fish model to test drug-induced alterations of locomotion and shoaling behaviour with a video-based analysis system. Consistent with its anxiolytic effects in zebrafish (Danio rerio), ethanol decreased shoal cohesion in anchovies. We also characterized the effects of drugs known to modulate the dopaminergic system in zebrafish and rodents. A D1 receptor agonist (SKF 38393) and a D1 receptor antagonist (SCH 23390) increased the time anchovy spent in the center of the arena, but neither drug had an impact on shoal cohesion. Finally, the D1 receptor agonist caused significantly lower meandering compared with fish treated with the D1 receptor antagonist and ethanol. This study suggests that anchovy is a suitable Caribbean marine model for toxicology studies.

Barott, KL, Barron ME, Tresguerres M.  2017.  Identification of a pH sensor in coral. Proceedings of the Royal Society B. 284:20171769.   10.1098/rspb.2017.1769   Abstract

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 pHsensor 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 acidificationwas 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.

Kwan, GT, Hamilton TJ, Tresguerres M.  2017.  CO2-induced ocean acidification does not affect individual or group behaviour in a temperate damselfish. Royal Society Open Science. 4   10.1098/rsos.170283   Abstract

Open ocean surface CO2 levels are projected to reach approximately 800 µatm, and ocean pH to decrease by approximately 0.3 units by the year 2100 due to anthropogenic CO2 emissions and the subsequent process of ocean acidification (OA). When exposed to these CO2/pH values, several fish species display abnormal behaviour in laboratory tests, an effect proposed to be linked to altered neuronal GABAA­ receptor function. Juvenile blacksmith (Chromis punctipinnis) are social fish that regularly experience CO2/pH fluctuations through kelp forest diurnal primary production and upwelling events, so we hypothesized that they might be resilient to OA. Blacksmiths were exposed to control conditions (pH ∼ 7.92; pCO2 ∼ 540 µatm), constant acidification (pH ∼ 7.71; pCO2 ∼ 921 µatm) and oscillating acidification (pH ∼ 7.91, pCO2 ∼ 560 µatm (day), pH ∼ 7.70, pCO2 ∼ 955 µatm (night)), and caught and tested in two seasons of the year when the ocean temperature was different: winter (16.5 ± 0.1°C) and summer (23.1 ± 0.1°C). Neither constant nor oscillating CO2-induced acidification affected blacksmith individual light/dark preference, inter-individual distance in a shoal or the shoal's response to a novel object, suggesting that blacksmiths are tolerant to projected future OA conditions. However, blacksmiths tested during the winter demonstrated significantly higher dark preference in the individual light/dark preference test, thus confirming season and/or water temperature as relevant factors to consider in behavioural tests.

Tresguerres, M, Hamilton TJ.  2017.  Acid-base physiology, neurobiology and behaviour in relation to CO2-induced ocean acidification. J Exp Biol. 220:2136-2148.   10.1242/jeb.144113   Abstract

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.

Hamilton, TJ, Tresguerres M, Kline DI.  2017.  Dopamine D1 receptor activation leads to object recognition memory in a coral reef fish. Biology Letters. 13:20170183.   10.1098/rsbl.2017.0183   AbstractWebsite

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.

Roa, JN, Tresguerres M.  2017.  Bicarbonate‐sensing soluble adenylyl cyclase is present in the cell cytoplasm and nucleus of multiple shark tissues. Physiological Reports. 5   10.14814/phy2.13090   Abstract

The enzyme soluble adenylyl cyclase (sAC) is directly stimulated by bicarbonate (HCO3−) to produce the signaling molecule cyclic adenosine monophosphate (cAMP). Because sAC and sAC‐related enzymes are found throughout phyla from cyanobacteria to mammals and they regulate cell physiology in response to internal and external changes in pH, CO2, and HCO3−, sAC is deemed an evolutionarily conserved acid‐base sensor. Previously, sAC has been reported in dogfish shark and round ray gill cells, where they sense and counteract blood alkalosis by regulating the activity of V‐type H+‐ ATPase. Here, we report the presence of sAC protein in gill, rectal gland, cornea, intestine, white muscle, and heart of leopard shark Triakis semifasciata. Co‐expression of sAC with transmembrane adenylyl cyclases supports the presence of cAMP signaling microdomains. Furthermore, immunohistochemistry on tissue sections, and western blots and cAMP‐activity assays on nucleus‐enriched fractions demonstrate the presence of sAC protein in and around nuclei. These results suggest that sAC modulates multiple physiological processes in shark cells, including nuclear functions.

Hamilton, TJ, Morril A, Lucas K, Gallup J, Harris M, Healey M, Pitman T, Schalomon M, Digweed S, Tresguerres M.  2017.  Establishing zebrafish as a model to study the anxiolytic effects of scopolamine. Scientific Reports. 7:15081.   10.1038/s41598-017-15374-w   AbstractWebsite

Scopolamine (hyoscine) is a muscarinic acetylcholine receptor antagonist that has traditionally been used to treat motion sickness in humans. However, studies investigating depressed and bipolar populations have found that scopolamine is also effective at reducing depression and anxiety symptoms. The potential anxiety-reducing (anxiolytic) effects of scopolamine could have great clinical implications for humans; however, rats and mice administered scopolamine showed increased anxiety in standard behavioural tests. This is in direct contrast to findings in humans, and complicates studies to elucidate the specific mechanisms of scopolamine action. The aim of this study was to assess the suitability of zebrafish as a model system to test anxiety-like compounds using scopolamine. Similar to humans, scopolamine acted as an anxiolytic in individual behavioural tests (novel approach test and novel tank diving test). The anxiolytic effect of scopolamine was dose dependent and biphasic, reaching maximum effect at 800 µM. Scopolamine (800 µM) also had an anxiolytic effect in a group behavioural test, as it significantly decreased their tendency to shoal. These results establish zebrafish as a model organism for studying the anxiolytic effects of scopolamine, its mechanisms of action and side effects.

Tresguerres, M, Barott K, Barron ME, Deheyn D, Kline D, Linsmayer LB.  2017.  Cell Biology of Reef-Building Corals: Ion Transport, Acid/Base Regulation, and Energy Metabolism. Acid-Base Balance and Nitrogen Excretion in Invertebrates. ( Weihrauch D, O'Donnell M, Eds.).:193-218.: Springer International Publishing   10.1007/978-3-319-39617-0_7   Abstract

Coral reefs are built by colonial cnidarians that establish a symbiotic relationship with dinoflagellate algae of the genus Symbiodinium. The processes of photosynthesis, calcification, and general metabolism require the transport of diverse ions across several cellular membranes and generate waste products that induce acid/base and oxidative stress. This chapter reviews the current knowledge on coral cell biology with a focus on ion transport and acid/base regulation while also discussing related aspects of coral energy metabolism.

Wilson, CM, Roa JN, Cox GK, Tresguerres M, Farrell AP.  2016.  Introducing a novel mechanism to control heart rate in the ancestral Pacific hagfish. Journal of Experimental Biology. 219:3227-3236.   10.1242/jeb.138198   AbstractWebsite

Although neural modulation of heart rate is well established among chordate animals, the Pacific hagfish (Eptatretus stoutii) lacks any cardiac innervation, yet it can increase its heart rate from the steady, depressed heart rate seen in prolonged anoxia to almost double its normal normoxic heart rate, an almost fourfold overall change during the 1-h recovery from anoxia. The present study sought mechanistic explanations for these regulatory changes in heart rate. We provide evidence for a bicarbonate-activated, soluble adenylyl cyclase (sAC)-dependent mechanism to control heart rate, a mechanism never previously implicated in chordate cardiac control.

Thomsen, J, Himmerkus N, Holland N, Sartoris FJ, Bleich M, Tresguerres M.  2016.  Ammonia excretion in mytilid mussels is facilitated by ciliary beating. Journal of Experimental Biology.   10.1242/jeb.139550   Abstract

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.

Roa, JN, Tresguerres M.  2016.  Soluble adenylyl cyclase is an acid-base sensor in epithelial base-secreting cells. Am J Physiol Cell Physiol. 311:C340-9.   10.1152/ajpcell.00089.2016   Abstract

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.

Tresguerres, M.  2016.  Novel and potential physiological roles of vacuolar-type H+-ATPase in marine organisms. J Exp Biol. 219:2088-97.   10.1242/jeb.128389   Abstract

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.

Hamilton, TJ, Kwan GT, Gallup J, Tresguerres M.  2016.  Acute fluoxetine exposure alters crab anxiety-like behaviour, but not aggressiveness. Scientific Reports. 6:19850.: Macmillan Publishers Limited   10.1038/srep19850   Abstract

Aggression and responsiveness to noxious stimuli are adaptable traits that are ubiquitous throughout the animal kingdom. Like vertebrate animals, some invertebrates have been shown to exhibit anxiety-like behaviour and altered levels of aggression that are modulated by the neurotransmitter serotonin. To investigate whether this influence of serotonin is conserved in crabs and whether these behaviours are sensitive to human antidepressant drugs; the striped shore crab, Pachygrapsus crassipes, was studied using anxiety (light/dark test) and aggression (mirror test) paradigms. Crabs were individually exposed to acute doses of the selective serotonin reuptake inhibitor, fluoxetine (5 or 25 mg/L), commonly known as Prozac®, followed by behavioural testing. The high dose of fluoxetine significantly decreased anxiety-like behaviour but had no impact on mobility or aggression. These results suggest that anxiety-like behaviour is more sensitive to modulation of serotonin than is aggressiveness in the shore crab.

Barott, K, Tresguerres M.  2015.  Immunolocalization of Proteins in Corals: the V-type H+-ATPase Proton Pump. Bio-protocol. 5 AbstractWebsite

Here we describe the immunolocalization of a membrane-bound proton pump, the V-type H+-ATPase (VHA), in tissues and isolated cells of scleractinian corals. Immunolocalization of coral proteins requires additional steps not required for various model organisms, such as decalcification of the coral skeleton for immunohistochemistry or removal of cells away from the skeleton for immunocytochemistry. The tissue and cell preparation techniques described here can be adapted for localization of other coral proteins, provided the appropriate validation steps have been taken for the primary antibodies and species of coral used. These techniques are important for improving our understanding of coral cell physiology

Barott, KL, Perez SO, Linsmayer LB, Tresguerres M.  2015.  Differential localization of ion transporters suggests distinct cellular mechanisms for calcification and photosynthesis between two coral species. American Journal of Physiology - Regulatory, Integrative and Comparative Physiology .   10.1152/ajpregu.00052.2015   Abstract

Ion transport is fundamental for multiple physiological processes, including but not limited to pH regulation, calcification, and photosynthesis. Here, we investigated ion-transporting processes in tissues from the corals Acropora yongei and Stylophora pistillata, representatives of the complex and robust clades that diverged over 250 million years ago. Antibodies against complex IV revealed that mitochondria, an essential source of ATP for energetically costly ion transporters, were abundant throughout the tissues of A. yongei. Additionally, transmission electron microscopy revealed septate junctions in all cell layers of A. yongei, as previously reported for S. pistillata, as well as evidence for transcellular vesicular transport in calicoblastic cells. Antibodies against the alpha subunit of Na(+)/K(+)-ATPase (NKA) and plasma membrane Ca(2+)-ATPase (PMCA) immunolabeled cells in the calicoblastic epithelium of both species, suggesting conserved roles in calcification. However, NKA was abundant in the apical membrane of the oral epithelium in A. yongei but not S. pistillata, while PMCA was abundant in the gastroderm of S. pistillata but not A. yongei. These differences indicate that these two coral species utilize distinct pathways to deliver ions to the sites of calcification and photosynthesis. Finally, antibodies against mammalian sodium bicarbonate cotransporters (NBC; SLC4 family) resulted in strong immunostaining in the apical membrane of oral epithelial cells and in calicoblastic cells in A. yongei, a pattern identical to NKA. Characterization of ion transport mechanisms is an essential step toward understanding the cellular mechanisms of coral physiology and will help predict how different coral species respond to environmental stress.

Barott, KL, Venn AA, Perez SO, Tambutte S, Tresguerres M.  2015.  Coral host cells acidify symbiotic algal microenvironment to promote photosynthesis. Proc Natl Acad Sci U S A. 112:607-12.   10.1073/pnas.1413483112   Abstract

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 approximately 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.