Role of carbonic anhydrase in the supply of inorganic carbon to the giant clam—zooxanthellate symbiosis

The mechanism whereby inorganic carbon (C_i) is acquired by the symbiotic association between the giant clam (Tridacna derasa) and zooxanthellae (Symbiodinium sp.) has been investigated. C_i in the haemolymph of the clam is in equilibrium with the surrounding sea water. The photosynthesis rate exhibited by the intact clam varies as a function of the C_i concentration in the clam haemolymph. The gill tissue contains high carbonic anhydrase activity which may be important in adjusting the C_i equilibrium between haemolymph and sea water. Zooxanthellae (Symbiodinium sp.) isolated from the clam mantle prefer CO₂ to HCO ₃ ^- as a source of inorganic carbon. The zooxanthellae have low levels of carbonic anhydrase on the external surface of the cell; however, mantle extracts display high carbonic anhydrase activity. Carbonic anhydrase is absent from the mantle of aposymbiotic clams (T. gigas), indicating that this enzyme may be essential to the symbiosis. The enzyme is probably associated with the zooxanthellae tubes in the mantle. The results indicate that carbonic anhydrase plays an important role in the supply of carbon dioxide within the clam symbiosis.     

Studies on enzymes associated with calcification of the cuticle of the hermit crab Clibanarius olivaceous

In the anomuran Clibanarius olivaceous Henderson, the cuticle of the cephalic and thoracic regions is calcified, while that of the abdominal region is uncalcified. Histochemical observations have been made on the possible enzymes involved in the process of calcification of cuticle. The calcified regions show the presence of enzymes such as phosphorylase, alkaline phosphatase and carbonic anhydrase. The possible role of phosphorylase in calcification may be production of phosphoric esters, which form the required substrate for alkaline phosphatase. The latter appear to be associated with the production of phosphate ions from phosphoric esters. The carbonic anhydrase may be associated with the production of carbonate ions. The suggestion that these enzymes are involved in the process of calcification is further supported by experimental evidence of inhibition of the action of enzymes by enzyme inhibitors. The inhibition of enzymes leads to reduction in the amount of calcium added to the cuticle.This paper formed part of a thesis approved for the award of Ph. D. degree of the University of Madras.     

Response of hydrothermal vent vestimentiferan <Emphasis Type="Italic">Riftia pachyptila</Emphasis> to differences in habitat chemistry

Vestimentiferan tubeworms, which rely on intracellular sulfide-oxidizing autotrophic bacteria for organic carbon, flourish at deep-sea hydrothermal vents despite the erratic nature of their habitat. To assess the degree to which differences in habitat chemistry (sulfide, pH/CO₂) might impact host and symbiont metabolic activity, Riftia pachyptila tubeworms were collected from habitats with low (H₂S < 0.0001 mM) and high (up to 0.7 mM) sulfide concentrations. The elemental sulfur content of the symbiont-containing trophosome organ was lower in specimens collected from the low-sulfide site. Symbiont abundance, RubisCO activity, and trophosome carbon fixation rates were not significantly different for individuals collected from low- versus high-sulfide habitats. Carbonic anhydrase activities were higher in the anterior gas exchange organs of R. pachyptila from the low-sulfide habitat. Despite large differences in habitat chemistry, symbiont abundance and autotrophic potential were consistent, while the host appears to tailor carbonic anhydrase activity to environmental CO₂ availability.     

External and internal carbonic anhydrases in Dunaliella species

Four species of Dunaliella and a carbonic anhydrase deficient mutant of Dunaliella tertiolecta (Butcher), HL 25/8, developed a process for concentrating dissolved inorganic carbon when adapted on air or low CO₂ for 24 h in the light. However, the external carbonic anhydrase activities in Dunaliella species were nil or low compared with those in Chlamydomonas reinhardtii (Danegard) or some Chlorella species. D. tertiolecta had low and about equal levels of external and internal carbonic anhydrase. D. parva (Lerche) and the D. tertiolecta mutant had negligible external carbonic anhydrase. D. viridis (Teodoresco) and D. salina (Teodoresco), high salt tolerant species, had high activities of intenal carbonic anhydrase but low levels of external carbonic anhydrase. Antiserum prepared against the 37 kDa peptide of extracellular carbonic anhydrase from Chlamydomonas reinhardtii was immunoreactive with a polypeptide of 30 kDa in D. tertiolecta and its salt sensitive external carbonic anhydrase (CA) mutant HL 25/8. External CA activity from D. tertiolecta was stimulated about three-fold, by including 0.5 M NaCl in the assay medium, while internal CA was not significantly affected by NaCl. External CA activities in the other species were insensitive to NaCl, while their internal activities were 90% inhibited by 0.5 M NaCl. Sorbitol only partly replaced NaCl in stimulating the external CA from D. tertiolecta. These experiments were performed with Dunaliella spp. grown in controlled laboratory cultures during September through November 1989.     

Photosynthetic carbon acquisition in the red alga Gracilaria conferta

In this continuing study on photosynthesis of the marine red alga Gracilaria conferta, it was found that ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) in crude extracts had a K _m (CO₂) of 85 M. Since seawater contains only ca. 10 M CO₂, it appears that this alga must possess a CO₂ concetrating system in order to supply sufficient CO₂ to the vicinity of the enzyme. Because this species is a C₃ plant (and thus lacks the C₄ system for concentrating CO₂), but can utilize HCO₃ ^- as an exogenous carbon source, we examined whether HCO₃ ^- uptake could be the initial step of such a CO₂ concetrating system. The surface pH of G. conferta thalli was 9.4 during photosynthesis. At this pH, estimated maximal uncatalyzed HCO₃ ^- dehydration (CO₂ formation) within the unstirred layer was too slow to account for measured phostosynthetic rates, even in the presence of an external carbonic anhydrase inhibitor. This observation, and the marked pH increase in the unstirred layer following the onset of light, suggests that a HCO₃ ^- transport system (probably coupled to transmembrane H⁺/OH^- fluxes) operates at the plasmalemma level. The involvement of surface-bound carbonic anhydrase in such a system remains, however, obscure. The apparent need of marine macroalgae such as G. conferta for CO₂ concentrating mechanisms is discussed with regard to their low affinity of Rubisco to CO₂ and the low rate of CO₂ supply in water. The close similarity between rates of Rubisco carboxylation and measured photosynthesis further suggests that the carboxylase activity, rather than inorganic carbon transport and intercoversion events, could be an internal limiting factor for photosynthetic rates of G. conferta.     

Alkaline phosphatase and carbonic anhydrase activity associated with arm regeneration in the seastarAsterias forbesi

A sharp increase in alkaline phosphatase (AP) activity occurred 7 d after arm autotomy inAsterias forbesi. This activity remained elevated at 20 d post-autotomy, during which time active ossicle-calcification was occurring. By 60 d post-autotomy, AP activity had returned to the level of non-regenerating individuals. Conversely, carbonic anhydrase (CA) activity exhibited no initial change in response to arm autotomy. By Day 60 a 50% increase in CA activity was observed. AP is localized in the outer membranes and associated microvilli of the coelomic epithelium and is not associated directly with the tissues involved in calcification. CA activity was highest in the tissues containing the ossicles, but was also present in the non-calcifying tube feet. These data suggest that inA. forbesi AP is involved in cell division and differentiation associated with wound-healing and the initiation of the regeneration process, and that CA may facilitate the ongoing maturation process, of newly formed ossicles.     

Strategies for carbon acquisition in the red marine macroalga Coccotylus truncatus from the Baltic Sea

Marine macroalgae need carbon-concentrating mechanisms because they have only limited access to CO₂ in their natural environment. Previous studies have shown that one important strategy common to many algae is the activity of periplasmic carbonic anhydrases that catalyse the dehydration of HCO₃^- into CO₂. The latter can then cross the plasma membrane by passive diffusion. We hypothesised that an active (energy-consuming) mechanism might also be involved in the membrane transport of CO₂, as is the case in a number of microalgae. Coccotylus truncatus was chosen as a model organism for this study because it belongs to a group of algae that usually lack direct HCO₃^- uptake: sublittoral red algae. The method used to study carbon uptake was pH drift of the seawater medium surrounding the algae in a closed vessel, with and without the addition of specific inhibitors or proton buffers. Measured parameters included pH, total inorganic carbon and alkalinity of the seawater medium. Our results suggest that, in C. truncatus, periplasmic carbonic anhydrase as well as H⁺ extrusion, probably driven by a vanadate-sensitive P-type H⁺-ATPase (proton pump), are involved in CO₂ uptake. No direct uptake of HCO₃^- was discovered. This paper also presents data on the buffer capacity of several proton buffers and the carbon-uptake inhibitors acetazolamide, 4,4'-diisothiocyano-stilbene-2,2'-disulfonate (DIDS) and orthovanadate in Baltic Sea water with a salinity of 6.5 psu.     

Physiological responses of the calcifying rhodophyte, <Emphasis Type="Italic">Corallina officinalis</Emphasis> (L.), to future CO<Subscript>2</Subscript> levels

Future atmospheric CO₂ levels will most likely have complex consequences for marine organisms, particulary photosynthetic calcifying organisms. Corallina officinalis L. is an erect calcifying macroalga found in the inter- and subtidal regions of temperate rocky coastlines and provides important substrate and refugia for marine meiofauna. The main goal of the current study was to determine the physiological responses of C. officinalis to increased CO₂ concentrations expected to occur within the next century and beyond. Our results show that growth and production of inorganic material decreased under high CO₂ levels, while carbonic anhydrase activity was stimulated and negatively correlated to algal inorganic content. Photosynthetic efficiency based on oxygen evolution was also negatively affected by increased CO₂. The results of this study indicate that C. officinalis may become less competitive under future CO₂ levels, which could result in structural changes in future temperate intertidal communities.     

The impact of iron limitation on the physiology of the Antarctic diatom Chaetoceros simplex

Iron availability strongly governs the growth of Southern Ocean phytoplankton. To investigate how iron limitation affects photosynthesis as well as the uptake of carbon and iron in the Antarctic diatom Chaetoceros simplex, a combination of chlorophyll a fluorescence measurements and radiotracer incubations in the presence and absence of chemical inhibitors was conducted. Iron limitation in C. simplex led to a decline in growth rates, photochemical efficiency and structural changes in photosystem II (PSII), including a reorganisation of photosynthetic units in PSII and an increase in size of the functional absorption cross section of PSII. Iron-limited cells further exhibited a reduced plastoquinone pool and decreased photosynthetic electron transport rate, while non-photochemical quenching and relative xanthophyll pigment content were strongly increased, suggesting a photoprotective response. Additionally, iron limitation resulted in a strong decline in carbon fixation and thus the particulate organic carbon quotas. Inhibitor studies demonstrated that, independent of the iron supply, carbon fixation was dependent on internal, but not on extracellular carbonic anhydrase activity. Orthovanadate more strongly inhibited iron uptake in iron-limited cells, indicating that P-type ATPase transporters are involved in iron uptake. The stronger reduction in iron uptake by ascorbate in iron-limited cells suggests that the re-oxidation of iron is required before it can be taken up and further supports the presence of a high-affinity iron transport pathway. The measured changes to photosystem architecture and shifts in carbon and iron uptake strategies in C. simplex as a result of iron limitation provide evidence for a complex interaction of these processes to balance the iron requirements for photosynthesis and carbon demand for sustained growth in iron-limited waters.     

Histological and histochemical changes in the gills of the yellowtail Seriola quinqueradiata exposed to the Raphidphycean flagellate Chattonella marina

The gills of the yellowtail Seriola quinqueradiata, exposed to Chattonella marina a red tide species, were examined histologically and histochemically. The pavement cells of primary lamellae were swollen in the exposed specimens. This presumably indicates that C. marina contains undetermined toxic substances. The number of mucous cells containing mucous decreased in proportion to the duration of exposure. The loss of the mucous substance in the cells is probably due to the ichthyotoxic stimulus caused by C. marina. A decrease of carbonic anhydrase activity in the secondary lamellae was confirmed, when mucous substances were lost in most mucous cells. Since the carbonic anhydrase usually exists in the epithelia of the secondary lamellae, it was presumably resolved by C. marina. The decrease of the carbonic anhydrase activity may cause certain physiological disadvantages to the fish exposed to C. marina.     

Effect of nitrogen supply on photosynthesis and carbonic anhydrase activity in the green seaweed Ulva rigida (Chlorophyta)

Photosynthesis rate and carbonic anhydrase (CA) activity have been studied in the green seaweed Ulva rigida C. Agardh (Chlorophyta) grown in seawater (SW) and SW supplemented with 40 M NH₄Cl (N-SW). Higher growth and maximal O₂ evolution rates were observed in N-SW- than in SW-grown sea-weeds. Western blot analysis of the total homogenates probed with antibodies raised against small subunits of ribulose-1,5-bisphosphate carboxylase/oxygenase (RuBisCO) showed crossreaction with a 15 kdalton polypeptide in both SW- and N-SW-grown plants, although the band was more intense in N-SW-grown plants. Carbonic anhydrase activities in the total homogenate and in the soluble protein fraction were higher in N-SW-grown plants. Although the pellets from both plants showed a considerable CA activity, the activity of CA in the thylakoid membranes was undetectable. The low nitrogen concentration is a major environmental factor that affects the level of RuBisCO and CA, and therefore CO₂ assimilation in U. rigida.     

Inorganic carbon uptake by photosynthetically active protoplasts of the red macroalga Chondrus crispus

Photosynthetically active protoplasts were isolated from Chondrus crispus Stackh. by treating thalli with -carrageenase produced from batch culture of Pseudomonas carrageenovora. Using the silicone oil centrifugation technique, it was found that the protoplasts: (1) did not generally accumulate inorganic carbon (C_i) above the concentration in their incubation medium; (2) were saturated at C_i concentrations of 3 to 4 mM; (3) had an intracellular pH of 7.50 when incubated at pH 7.5; and (4) their initial carbon fixation rate was reduced by carbonic anhydrase inhibitors. Although the carbon fixation rate of the protoplasts was about 30% that of thallus fragments, presumably due to the relatively harsh protoplast isolation treatment, the behavior of the protoplasts was similar to that of fragments. This similarity indicates that the protoplasts are photosynthetically active and behave as thallus fragments. Further, the data are consistent with the hypothesis that C. crispus acquires C_i for photosynthesis by the diffusion of CO₂ across the plasma membrane.     

Isolated bacteriocyte cell suspensions from the hydrothermal-vent tubeworm Riftia pachyptila,a potent tool for cellular physiology in a chemoautotrophic symbiosis

The hydrothermal-vent tubeworm Riftia pachyptila relies entirely on its intracellular chemoautotrophic symbionts to sustain its metabolism. The host must therefore provide them with inorganic metabolites, including carbon. This study describes a tool for studying cell processes occurring in a bacteria-containing cell by the dissociation of trophosome cell types. The physiological assays performed on cell preparations focused on carbon dioxide conversion and transport processes. Trophosome tissue was mechanically dissociated, resulting in cell suspensions enriched in small (7-20 µm) bacteriocytes, which were viable for several hours. In addition, medium-term cell cultures were also attempted. As a start to the understanding of the CO₂ metabolism of these cells, we were interested in evidence of carbonic anhydrase (CA) isoforms, ATPases and chloride exchangers. Variations in intracellular and extracellular pH, and in intracellular concentrations of sodium, potassium and chloride, were followed after addition of selective inhibitors. The data presented here suggest the occurrence of potential cytosolic and membrane-associated carbonic anhydrase isoforms in the bacteriocytes, proton-driven sodium-ATPases and a well represented anion transporter exchanging intracellular chloride against extracellular anions.     

Effect of inhibitors of carbonic anhydrase activity on photosynthesis in the red alga Soliera filiformis (Gigartinales: Rhodophyta)

The effect of light intensity, pH and carbonic anhydrase (CA) inhibitors on photosynthesis of the red marine macroalgae Solieria filiformis (Kützing) Gabrielson, collected from Taliarte (Gran Canaria, Canary Islands) in 1991, has been investigated. Plants taken from the sea (wild phenotype) developed spherical morphology (ball phenotype) after 2 mo culture in aerated tanks. The photosynthetic oxygen evolution in the wild phenotype was saturated at 100 mol photons m^-2s^-1, while the ball phenotype displayed saturation at 200 mol photons m^-2s^-1. The inhibitors of total CA activity (6-ethoxizolamide) and extracellular CA activity (dextran-bound sulfonamide) inhibited photosynthesis at pH 8.2, to 90 and 50% respectively, in both phenotypes. No inhibition of the photosynthetic oxygen evolution was detected at pH 6.5. CA activity was associated with both supernatant and pellet fractions of crude extracts of S. filiformis. The rate of alkalization of the medium by the algae was dependent on light intensity. We suggest that carbon dioxide is the general form of inorganic carbon transported across the plasmamembrane in S. filiformis. HCO₃ transport into the cell takes place simultaneously by an indirect mechanism (dehydration to CO₂ catalyzed by CAext) and by direct uptake. Extracellular (CAext) and intracellular (CAint) CAs are involved in the mechanisms of inorganic carbon assimilation by S. filiformis.     

Determination of canthaxanthin in the red coral (<Emphasis Type="Italic">Corallium rubrum</Emphasis>) from Marseille by HPLC combined with UV and MS detection

Corallium rubrum is the most famous and precious coral due to the intense characteristic red colour of its skeleton. We have determined the presence of carotenoids in natural samples of C. rubrum from Marseille, Riou, and investigated their chemical composition. Analysis was performed both on soft tissues and hard tissues including spicules and skeleton. Since hard tissues are made of a mineral fraction and an organic fraction obtained after demineralization, extraction was performed both with and without demineralization by EDTA. The extracts were analyzed by complementary methods of TLC, HPLC/DAD and HPLC/MS. The components were separated by RP-18 chromatography column using acetonitrile/methanol for HPLC/DAD and acetonitrile/water gradient for HPLC/MS analysis. Our results give the first evidence of the presence of canthaxanthin, 4,4′-diketo-β-carotene, as the major carotenoid in all samples. Spicules exhibited higher content in carotenoids than skeleton. Demineralization treatment improved the efficiency of carotenoid extraction by an average factor of 5 and showed that organic matrix contains canthaxanthin.     

Physiological role of branchial carbonic anhydrase in the shore crabCarcinus maenas

Excretion of total CO₂ and uptake of sodium and chloride ions across the branchial epithelium of the posterior gills of the shore crabCarcinus maenas, collected from Kiel Bay (Baltic Sea) in 1989, were measured using isolated perfused gill preparations. Total CO₂ effluxes depended on the HCO ₃ ^- concentration of the internal perfusate in a saturable mode and were inhibited by internally and externally applied acetazolamide at 10^–4 M. Potential differences between hemolymph space and medium did not change significantly during experimental treatments. Neither a bicarbonate gradient (6 mM) directed from the internal perfusate to external bath solution nor symmetrically applied 10^–4 M acetazolamide significantly influenced the influxes of Na⁺ and Cl^–. Results confirmed the role of carbonic anhydrase in CO₂ excretion but called into question the assumed functioning of the enzyme in branchial ion transport processes.     

Biomineralization markers during early shell formation in the European abalone <Emphasis Type="Italic">Haliotis tuberculata</Emphasis>, Linnaeus

Larval shell formation was investigated in the European abalone Haliotis tuberculata. Stages of mineralization as well as enzymatic and endocrine biomarkers were monitored throughout larval development, from hatching to post-larval stages. Polarized light microscopy and infrared spectroscopy analyses revealed the presence of crystallized calcium carbonate arranged in aragonite polymorphs from the late trochophore stage. A correlation between the main steps of shell formation and enzymatic activities of alkaline phosphatase and carbonic anhydrase was seen. The variations of these biologic activities were related to the onset of mineralization, the rapid shell growth, and the switch from larval to juvenile shell following metamorphosis. Furthermore, a strong increase in the level of calcitonin gene-related molecules was measured in post-larvae, suggesting that endocrine control takes place after metamorphosis. The changes measured for the three biomineralization markers together with mineralogical analysis allowed us to correlate physiologic mechanisms with early steps of abalone shell formation.