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2017
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   AbstractWebsite

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.

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.

2016
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.

2015
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.

Clifford, AM, Goss GG, Roa JN, Tresguerres M.  2015.  Acid/base and ionic regulation in hagfish. Hagfish biology. ( Edwards SL, Goss GG, Eds.).:277-298., Boca Raton, FL: CRC Press
2014
Roa, JN, Munevar CL, Tresguerres M.  2014.  Feeding induces translocation of vacuolar proton ATPase and pendrin to the membrane of leopard shark (Triakis semifasciata) mitochondrion-rich gill cells. Comp Biochem Physiol A Mol Integr Physiol. 174:29-37.   10.1016/j.cbpa.2014.04.003   Abstract

In this study we characterized mitochondrion-rich (MR) cells and regulation of acid/base (A/B) relevant ion-transporting proteins in leopard shark (Triakis semifasciata) gills. Immunohistochemistry revealed that leopard shark gills posses two separate cell populations that abundantly express either Na(+)/K(+)-ATPase (NKA) or V-H(+)-ATPase (VHA), but not both ATPases together. Co-immunolocalization with mitochondrial Complex IV demonstrated, for the first time in shark gills, that both NKA- and VHA-rich cells are also MR cells, and that all MR cells are either NKA- or VHA-rich cells. Additionally we localized the anion exchanger pendrin to VHA-rich cells, but not NKA-rich cells. In starved sharks, VHA was localized throughout the cell cytoplasm and pendrin was present at the apical pole (but not in the membrane). However, in a significant number of gill cells from fed leopard sharks, VHA translocated to the basolateral membrane (as previously described in dogfish), and pendrin translocated to the apical membrane. Our results highlight the importance of translocation of ion-transporting proteins to the cell membrane as a regulatory mechanism for A/B regulation.

Hamilton, TJ, Paz-Yepes J, Morrison RA, Palenik B, Tresguerres M.  2014.  Exposure to bloom-like concentrations of two marine Synechococcus cyanobacteria (strains CC9311 and CC9902) differentially alters fish behaviour. Conservation Physiology. 2   10.1093/conphys/cou020   AbstractWebsite

Coastal California experiences large-scale blooms of Synechococcus cyanobacteria, which are predicted to become more prevalent by the end of the 21st century as a result of global climate change. This study investigated whether exposure to bloom-like concentrations of two Synechococcus strains, CC9311 and CC9902, alters fish behaviour. Black perch (Embiotoca jacksoni) were exposed to Synechococcus strain CC9311 or CC9902 (1.5 × 106 cells ml−1) or to control seawater in experimental aquaria for 3 days. Fish movement inside a testing arena was then recorded and analysed using video camera-based motion-tracking software. Compared with control fish, fish exposed to CC9311 demonstrated a significant preference for the dark zone of the tank in the light–dark test, which is an indication of increased anxiety. Furthermore, fish exposed to CC9311 also had a statistically significant decrease in velocity and increase in immobility and they meandered more in comparison to control fish. There was a similar trend in velocity, immobility and meandering in fish exposed to CC9902, but there were no significant differences in behaviour or locomotion between this group and control fish. Identical results were obtained with a second batch of fish. Additionally, in this second trial we also investigated whether fish would recover after a 3 day period in seawater without cyanobacteria. Indeed, there were no longer any significant differences in behaviour among treatments, demonstrating that the sp. CC9311-induced alteration of behaviour is reversible. These results demonstrate that blooms of specific marine Synechococcus strains can induce differential sublethal effects in fish, namely alterations light–dark preference behaviour and motility.

Tresguerres, M.  2014.  sAC from aquatic organisms as a model to study the evolution of acid/base sensing. Biochim Biophys Acta.   10.1016/j.bbadis.2014.06.021   Abstract

Soluble adenylyl cyclase (sAC) is poised to play multiple physiological roles as an acid/base (A/B) sensor in aquatic organisms. Many of these roles are probably similar to those in mammals; a striking example is the evolutionary conservation of a mechanism involving sAC, carbonic anhydrase and vacuolar H+-ATPase that acts as a sensor system and regulator of extracellular A/B in shark gills and mammalian epididymis and kidney. Additionally, the aquatic environment presents unique A/B and physiological challenges; therefore, sACs from aquatic organisms have likely evolved distinct kinetic properties as well as distinct physiological roles. sACs from aquatic organisms offer an excellent opportunity for studying the evolution of A/B sensing at both the molecular and whole organism levels. Moreover, this information could help understand and predict organismal responses to environmental stress based on mechanistic models.This article is part of a Special Issue entitled "The Role of Soluble Adenylyl Cyclase in Health and Disease," guest edited by J. Buck and L. R. Levin.

Tresguerres, M, Barott KL, Barron ME, Roa JN.  2014.  Established and potential physiological roles of bicarbonate-sensing soluble adenylyl cyclase (sAC) in aquatic animals. Journal of Experimental Biology. 217:663-672.   10.1242/jeb.086157   AbstractWebsite

Soluble adenylyl cyclase (sAC) is a recently recognized source of the signaling molecule cyclic AMP (cAMP) that is genetically and biochemically distinct from the classic G-protein-regulated transmembrane adenylyl cyclases (tmACs). Mammalian sAC is distributed throughout the cytoplasm and it may be present in the nucleus and inside mitochondria. sAC activity is directly stimulated by HCO3-, and sAC has been confirmed to be a HCO3- sensor in a variety of mammalian cell types. In addition, sAC can functionally associate with carbonic anhydrases to act as a de facto sensor of pH and CO2. The two catalytic domains of sAC are related to HCO3--regulated adenylyl cyclases from cyanobacteria, suggesting the cAMP pathway is an evolutionarily conserved mechanism for sensing CO2 levels and/or acid/base conditions. Reports of sAC in aquatic animals are still limited but are rapidly accumulating. In shark gills, sAC senses blood alkalosis and triggers compensatory H+ absorption. In the intestine of bony fishes, sAC modulates NaCl and water absorption. And in sea urchin sperm, sAC may participate in the initiation of flagellar movement and in the acrosome reaction. Bioinformatics and RT-PCR results reveal that sAC orthologs are present in most animal phyla. This review summarizes the current knowledge on the physiological roles of sAC in aquatic animals and suggests additional functions in which sAC may be involved.

Hamilton, TJ, Holcombe A, Tresguerres M.  2014.  CO2-induced ocean acidification increases anxiety in Rockfish via alteration of GABAA receptor functioning. Proc Biol Sci. 281:20132509.   10.1098/rspb.2013.2509   Abstract

The average surface pH of the ocean is dropping at a rapid rate due to the dissolution of anthropogenic CO2, raising concerns for marine life. Additionally, some coastal areas periodically experience upwelling of CO2-enriched water with reduced pH. Previous research has demonstrated ocean acidification (OA)-induced changes in behavioural and sensory systems including olfaction, which is due to altered function of neural gamma-aminobutyric acid type A (GABAA) receptors. Here, we used a camera-based tracking software system to examine whether OA-dependent changes in GABAA receptors affect anxiety in juvenile Californian rockfish (Sebastes diploproa). Anxiety was estimated using behavioural tests that measure light/dark preference (scototaxis) and proximity to an object. After one week in OA conditions projected for the next century in the California shore (1125 +/- 100 microatm, pH 7.75), anxiety was significantly increased relative to controls (483 +/- 40 microatm CO2, pH 8.1). The GABAA-receptor agonist muscimol, but not the antagonist gabazine, caused a significant increase in anxiety consistent with altered Cl(-) flux in OA-exposed fish. OA-exposed fish remained more anxious even after 7 days back in control seawater; however, they resumed their normal behaviour by day 12. These results show that OA could severely alter rockfish behaviour; however, this effect is reversible.

2013
Jew, CJ, Wegner NC, Yanagitsuru Y, Tresguerres M, Graham JB.  2013.  Atmospheric oxygen levels affect mudskipper terrestrial performance: implications for early tetrapods. Integr Comp Biol. 53:248-57.   10.1093/icb/ict034   Abstract

The Japanese mudskipper (Periophthalmus modestus), an amphibious fish that possesses many respiratory and locomotive specializations for sojourns onto land, was used as a model to study how changing atmospheric oxygen concentrations during the middle and late Paleozoic Era (400-250 million years ago) may have influenced the emergence and subsequent radiation of the first tetrapods. The effects of different atmospheric oxygen concentrations (hyperoxia = 35%, normoxia = 21%, and hypoxia = 7% O2) on terrestrial performance were tested during exercise on a terrestrial treadmill and during recovery from exhaustive exercise. Endurance and elevated post-exercise oxygen consumption (EPOC; the immediate O2 debt repaid post-exercise) correlated with atmospheric oxygen concentration indicating that when additional oxygen is available P. modestus can increase oxygen utilization both during and following exercise. The time required post-exercise for mudskippers to return to a resting metabolic rate did not differ between treatments. However, in normoxia, oxygen consumption increased above hyperoxic values 13-20 h post-exercise suggesting a delayed repayment of the incurred oxygen debt. Finally, following exercise, ventilatory movements associated with buccopharyngeal aerial respiration returned to their rest-like pattern more quickly at higher concentrations of oxygen. Taken together, the results of this study show that P. modestus can exercise longer and recover quicker under higher oxygen concentrations. Similarities between P. modestus and early tetrapods suggest that increasing atmospheric oxygen levels during the middle and late Paleozoic allowed for elevated aerobic capacity and improved terrestrial performance, and likely led to an accelerated diversification and expansion of vertebrate life into the terrestrial biosphere.

Tresguerres, M, Katz S, Rouse GW.  2013.  How to get into bones: proton pump and carbonic anhydrase in Osedax boneworms. Proceedings of the Royal Society B-Biological Sciences. 280   10.1098/rspb.2013.0625   AbstractWebsite

Osedax are gutless siboglinid worms that thrive on vertebrate bones lying on the ocean floor, mainly those of whales. The posterior body of female Osedax penetrates into the bone forming extensions known as 'roots', which host heterotrophic symbiotic bacteria in bacteriocytes beneath the epidermis. The Osedax root epithelium presumably absorbs bone collagen and/or lipids, which are metabolized by the symbiotic bacteria that in turn serve for Osedax's nutrition. Here, we show that Osedax roots express extremely high amounts of vacuolar-H+-ATPase (VHA), which is located in the apical membrane and in cytoplasmic vesicles of root and ovisac epithelial cells. The enzyme carbonic anhydrase (CA), which catalyses the hydration of CO2 into H+ and HCO3-, is also expressed in roots and throughout Osedax body. These results suggest Osedax roots have massive acid-secreting capacity via VHA, fuelled by H+ derived from the CA-catalysed hydration of CO2 produced by aerobic metabolism. We propose the secreted acid dissolves the bone carbonate matrix to then allow the absorption of bone-derived nutrients across the skin. In an exciting example of convergent evolution, this model for acid secretion is remarkably similar to mammalian osteoclast cells. However, while osteoclasts dissolve bone for repairing and remodelling, the Osedax root epithelium secretes acid to dissolve foreign bone to access nutrients.

Barott, KL, Helman Y, Haramaty L, Barron ME, Hess KC, Buck J, Levin LR, Tresguerres M.  2013.  High adenylyl cyclase activity and in vivo cAMP fluctuations in corals suggest central physiological role. Sci. Rep.. 3: Macmillan Publishers Limited. All rights reserved   10.1038/srep01379   AbstractWebsite

Corals are an ecologically and evolutionarily significant group, providing the framework for coral reef biodiversity while representing one of the most basal of metazoan phyla. However, little is known about fundamental signaling pathways in corals. Here we investigate the dynamics of cAMP, a conserved signaling molecule that can regulate virtually every physiological process. Bioinformatics revealed corals have both transmembrane and soluble adenylyl cyclases (AC). Endogenous cAMP levels in live corals followed a potential diel cycle, as they were higher during the day compared to the middle of the night. Coral homogenates exhibited some of the highest cAMP production rates ever to be recorded in any organism; this activity was inhibited by calcium ions and stimulated by bicarbonate. In contrast, zooxanthellae or mucus had >1000-fold lower AC activity. These results suggest that cAMP is an important regulator of coral physiology, especially in response to light, acid/base disturbances and inorganic carbon levels.