Characterization and role of carbonic anhydrase in the calcification process of the azooxanthellate coral <Emphasis Type="Italic">Tubastrea aurea</Emphasis>

In zooxanthellate corals, the photosynthetic fixation of carbon dioxide and the precipitation of CaCO₃ are intimately linked both spatially and temporally making it difficult to study carbon transport mechanisms involved in each pathway. When studying Tubastrea aurea, a coral devoid of zooxanthellae, we can focus on carbon transport mechanisms involved only in the calcification process. We performed this study to characterize T. aurea carbonic anhydrase and to determine its role in the calcification process. We have shown that inhibition of tissular carbonic anhydrase activity affects the calcification rate. We have measured the activity of this enzyme both in the tissues and in the organix matrix extracted from the skeleton. Our results indicate that organic matrix proteins, which are synthesized by the calcifying tissues, are not only structural proteins, but they also play a crucial catalytic role by eliminating the kinetic barrier to interconversion of inorganic carbon at the calcification site. By immunochemistry we have demonstrated the presence of a protein both in the tissues and in the organic matrix, which shares common features with prokaryotic carbonic anhydrases.     

The distribution of zinc in the oyster Ostrea edulis and its relation to enzymic activity and to other metals

The role of zinc in the oyster Ostrea edulis Linnaeus has been studied in its relation to the zinc-dependent enzymes present and in relation to the copper, calcium, magnesium, sodium, potassium and phosphate contents. Only carbonic anhydrase, alkaline phosphatase, carboxypeptidase A and malic dehydrogenase zinc metalloenzyme activities could be detected. -D-mannosidase, a zinc-dependent enzyme hitherto not reported for the oyster, was also detected. After tissue dissection into muscle, palps, gills, mantle and digestive mass and subcellular fractionation of these tissues, analysis indicated that no single tissue concentrates zinc or the zinc-dependent enzymes. The total amount of zinc found is far in excess of the amount of zinc contributed by the zinc-dependent enzymes, but the amount of non-dialysable zinc is of the same order of magnitude. It is suggested that this apparent excess of dialysable zinc is a consequence of the high levels of calcium found in the tissues, demonstrating a competition between calcium and zinc in their uptake, as is well documented in many other phyla.     

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.     

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.     

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.     

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.     

Enzymatic and cellular level effects of chromium∗

Exposure of Cr to four hepatic enzymes activities of tilapia, viz. acid‐ and alkaline phosphatases, catalase and glucose‐6‐phosphatase, was contrary to the results obtained for Cd and Ni. This is the only heavy metal investigated thus far that dramatically augmented glucose‐6‐phosphatase by approximately 83% at a concentration of 12 mg L^‐1 in vivo in lieu of the fact of an initial inhibition of approximately 45 % at a lower concentration of Cr; 6 mg L^‐1. In the case of acid phosphatase and catalase activities progressive increment was observed up to 50 and 217% respectively at a concentration of 12 mg L^‐1 Cr. On the other hand, in contrast to all the investigated enzymes interestingly alkaline phosphatase was inhibited continuously at all concentrations up to 46% at 12 mgL^‐1 Cr. In vitro experiments were contrary to the above mentioned results, whereby all hepatic enzymes were inhibited with major inhibition observed for acid phosphatase of approximately 60% from 5 mgL^‐1 Cr onwards in the system.

At cellular level, Cr exposure at a lethal dose of 12 mgL^‐1 demonstrated similar effects to that of Cd. In general, the glycogen and fat reserves were depleted while lysosomal activity is increased. As compared to the effects of Cd, the mitochondria did not indicate any prominent reflection in the formation of intramitochondrial bodies. Further, similar to Cd, the cell membrane as well as nucleus were not affected.     

Acid and alkaline phosphatase activities in the clam Ruditapes philippinarum

Acid and alkaline phosphatase activities have been partially characterized in Ruditapes philippinarum (Adams and Reeve, 1850). Two activity peaks at pH=4.5 and pH 10.5 were detected in the gill, digestive gland, mantle, siphon and foot. Acid phosphatase activity was higher than that of alkaline phosphatase. The highest activity for both enzymes was observed in the digestive gland and, in decreasing order, the gill, foot, siphon and mantle. Alkaline phosphatase activity was similar in the mantle, siphon and foot. K _m values were determined for both enzymes in the gill and digestive gland. Hill coefficients were near 1, indicating no allosteric behaviour for either enzyme in the two organs. The optimum temperature was the same for acid phosphatase in both gill and digestive gland (50 °C), while for alkaline phosphatase it differed for these two organs (gill, 40 °C; digestive gland, 35 °C). The apparent activation energy was obtained from Arrhenius plots, and ranged from 8.61 kcal/mol for alkaline phosphatase in the gill, to 10.84 kcal/mol for acid phosphatase in the digestive gland. The effects of metals (1 mM conc) on both enzyme activities were assayed in vitro. Hg strongly inhibited the enzyme activities in the gill and digestive gland, probably because of its affinity for the sulphydryl group. Histochemically, acid phosphatase in the gill was located in a granular form throughout the gill cells, but was undetectable in the ciliate epithelium of the gill filaments. Alkaline phosphatase was located in the gill skeleton. Clam size and phosphatase activities were inversely related, probably reflecting a decrease in shell deposition with inereasing size. As a function of season, both enzymes were present in lowest amounts in winter, when undifferentiated sex cells were predominant in the germinative epithelium, and highest in summer, when ripe individuals of both sexes were more frequent.     

The water economy of the sea grass Thalassia testudinum

The osmotic values in the leaves of Thalassia testudinum Banks ex König, collected from the natural habitat, were found to be 32 atm; the relative transpiration was 100%. Electronmicroscopical analysis of the cuticle reveals many perforations. The critical sublethal water deficit lies near 65%. In dense meadows the green surface area of the plants amounts to 18.56 m²/m² bottom area.     

Enzymatic and cellular level effects of nickel∗

The effects of Ni on hepatic enzymes of tilapia, viz. acid‐ and alkaline phosphatases, catalase and glucose‐6‐phosphatase, both under in vivo and in vitro conditions reflected the following tendencies. In vivo conditions indicated maximal increase in activity for acid phosphatase at 3.00 ppm, equivalent to 28.5%, followed by a slight decrease and increase thereafter. As for alkaline phosphatase, gradual increase in activity was observed with maximal activity at 9 ppm of Ni, equivalent to 16.8%. Catalase demonstrated similar tendencies with maximal activity at 9.0 ppm, equivalent to 101.2%. In the case of glucose‐6‐phosphatase, the tendency was the reverse with maximal inhibition at 9.00 ppm, i.e. 41.9%. In contrast to in vivo conditions, in vitro systems indicated that all investigated enzymes were inhibited in the region of 4–10% except for catalase which demonstrated a slight increase by 5–6% in activity between concentrations of 10–15 ppm of Ni but thereafter continuous inhibitory effects prevailed.

At cellular level, exposure of tilapia to a lethal dose of 9 ppm of Ni indicated not much of an adverse effect except for a slight depletion in fat and glycogen content. In the case of mitochondria, they were normal and a few large secondary lysosomes were observed. In relation to the cell membrane no dramatic change was detected.     

Biochemical characterization of purified carbonic anhydrase from the octocoral Leptogorgia virgulata

Carbonic anhydrase (CA) has been isolated and purified from the octocoral Leptogorgia virgulata (Lamarck) in an effort to investigate its role in the mineralization and demineralization of spicules and other calcified hard tissues. Affinity-chromatography using Prontosil-derivatized carboxymethylcellulose (CM) Bio-Gel A provided a one-step purification for 30 kdalton polypeptides with carbonic anhydrase activity. Four distinct polypeptides (designated , , , and ) are separated from one another at this molecular weight by two-dimensional gel electrophoresis. The specific activity of the L. virgulata CA is on average 57.5±1.5 U g^-1, is inhibitable by 10^-6 M acetazolamide, and is unaffected by 5mM dithiotheitol. The amino acid composition of these polypeptides is similar to that of mammal, bird, reptile, fish and arthropod species. Antiserum made against the L. virgulata CA reacts specifically with the 30 kdalton polypeptides in western blots, and crossreacts with human CA I and II. Antiserum against avian CA II crossreacts with the L. virgulata 30 kdalton polypeptides. This is the first report of the characterization of a purified CA from an octocoral, and production of a CA antiserum to a species in the phylum Cnidaria.     

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.     

Effects of calcium,strontium, and magnesium on the coccolithophorid Cricosphaera (Hymenomonas) carterae. I. Calcification

The present investigation concerns the effects of calcium, strontium, and magnesium on calcification and mineralogy of the calcified bodies (coccoliths) of the coccolithophorid Cricosphaera (Hymenomonas) carterae. The capacity of cells to calcify in various concentrations of these ions was examined following preliminary decalcification in CO₂. At a concentration of 10^-2 M Ca, 75% of the cells formed coccoliths within 24 h and almost all cells were recalcified after 2 days. At 10^-3 or 10^-4 M Ca no recalcification occurred. However, with the addition of Sr to the Ca-deficient media, calcification took place as shown by observations of coccoliths and by analysis of Ca. The percentage of calcified cells increased with increasing concentrations of Sr. Strontium added to a Ca-deficient media was much more effective than an equivalent concentration of Ca. No Sr was deposited in the coccoliths. X-ray analysis demonstrated that calcite was deposited by cells in all concentrations of Ca and Sr at which calcification took place. At concentrations of Mg in the media from O. O to 4.2×10^-2 M, the cells retained their ability to calcify, although calcification was markedly reduced in the absence of Mg. In low Mg concentrations (1.3×10^-4 and 4.2×10^-6 M), the coccoliths were 60% calcite and 40% aragonite, and in teh absence of Mg, only calcite was formed.     

Effects of rogar and endosulfan on the metabolism of fresh water sponge (Spongilla lacustris)

Spongilla lacustris were exposed to sub lethal concentrations of pesticides, rogar and endosulfan for one month period. Metabolites like carbohydrates, protein and enzymes like those that peroxidase and carbonic anhydrase were estimated in the experimental and control animals. The results show, depletion of carbohydrates while protein elevated as the days progressed. Similarly an enzyme activity found to be decreased in exposed Spongilla lacustris.     

Characterization of alkaline phosphatase and organic phosphorous utilization in the oceanic dinoflagellate Pyrocystis noctiluca

Phosphate depleted Pyrocystis noctiluca (Murray) Schuett 1895 has at least one phosphomonoesterase (EC 3:1:3:1) which is triphasic between 0.1 and 222 mol P. The enzyme has a broad temperature range with maximum activity at 50 °C and a Q₁₀ of 1.4 to 1.5. A break in the Arrhenius plot at 35 °C implies the enzyme is membrane-bound. Cytological staining of whole cells and cell fractionation studies (showing 26 times higher specific activity in the particulate compared with the cytoplasmic fraction) suggest the enzyme is plasmalemma-bound. The enzyme has an absolute metal requirement which would be satisfied by Mg⁺⁺ but not Mn⁺⁺, Zn⁺⁺, Fe⁺⁺, or Co⁺⁺ at seawater concentrations. Alkaline phosphatase is a stable enzyme whose activity is not altered by inhibitors of protein synthesis. Orthophosphate inhibition of enzyme activity was largely eliminated in the presence of these inhibitors. Apparently, a protein induced by PO₄ ^3-, rather than PO₄ ^3- itself, inhibits alkaline phosphatase. Cell-free alkaline phosphatase can hydrolyze a variety of phosphate esters and linear polymers of inorganic phosphorus as well as disolved organic phosphorus from tropical oceanic waters. These same hydrolysable organic and inorganic phosphorus compounds support the axenic culture growth of P. noctiluca, suggesting that naturally occurring hydrolysable organic phosphorus compounds may also support the growth of this alga.     

Effects of pesticides on soil enzymes: a review

The use of pesticides in agriculture has highly increased during the last 40 years to increase crop yields. However, today most pesticides are polluting water, soil, atmosphere and food. Pesticides are also impact soil enzymes, which are essential catalysts ruling the quality of soil life. In particular, the activity of soil enzymes control nutrient cycles, and, in turn, fertilization. Here, we review the effects of pesticides on the activity of soil enzymes in terrestrial ecosystems. Enzymes include dehydrogenase, fluorescein diacetate hydrolase, acid phosphatase, alkaline phosphatase, phosphatase, β-glucosidase, cellulase, urease and aryl-sulfatase. Those enzymes are involved in the cycles of carbon, nitrogen, sulfur and phosphorus. The main points of our analysis are (1) the common inhibition of dehydrogenase in 61 % of studies, stimulation of cellulase in 56 % of studies and no response of aryl-sulfatase in 67 % of studies. (2) Fungicides have mainly negative effects on enzymatic activities. (3) Insecticides can be classified into two groups, the first group represented by endosulfan having an overall positive impact while the second group having a negative effect. (4) Herbicides can be classified into two groups, one group with few positive effect and another group with negative effect.     

Nature and composition of the cephalic and thoracic cuticles of the hermit crab Clibanarius olivaceous

The structure and composition of the cephalic and thoracic cuticles of Clibanarius olivaceous Henderson are described. The cephalic cuticle is completely hardened due to tanning of the epicuticle and the outer part of the procuticle (pigmented Layer). The thoracic cuticle is partially hardened. The differences in extent of tanning reflect the chemical composition of the respective regions. The epicuticle of the cephalic region contains a protein rich in tyrosine, which combines with a sterol to form a lipoprotein complex. This in turn forms the precursor to sclerotin. Phenolic substances appear to be oxidized to quinones by the action of phenolase. The outer part of the procuticle of the cephalic region also contains phenolic substances, but this layer remains only partially tanned. This partial hardening is due to the presence of an inhibitory substance having the nature of ascorbic acid. In the epicuticle of the thoracic region, however, an arrest in tanning appears to be due not to the presence of an inhibitory substance, but to the absence of phenolase necessary for the oxidation of tanning material. In C. olivaceous, the tanning process is much abbreviated. The primary cause of hardening is calcification. The sulphydryl groups and acid mucopolysaccharide detected in the cephalic and thoracic cuticles of C. olivaceous are involved in calcification. It is suggested that hardening of the cephalic and thoracic cuticles in C. olivaceous constitutes a protection during those periods when these parts are exposed to the environment.     

Mating expenditures reduced via female sex pheromone modulation in the primitively eusocial halictine bee, Lasioglossum (Evylaeus) malachurum (Hymenoptera: Halictidae)

The present paper reports on behavioral experiments and gas chromatographic analysis of chemical communication in the mating biology of the primitively eusocial sweat bee Lasioglossum (Evylaeus) malachurum. In a dual-choice experiment, a female made odorless was significantly less attractive than an untreated one. Attraction in L. (Evylaeus) malachurum is therefore mediated by a female-produced sex pheromone. Further bioassays showed that unmated gynes are more attractive to males than mated ones. Males are able to differentiate between the two groups of females as little as 3 h after mating. Biotests with different samples obtained from attractive gynes showed surface extracts to be most attractive. Behavioral tests with synthetic copies of the compounds identified as cuticular constituents of virgin gynes were highly attractive to males; the volatile bouquets consisting of n-alkanes, n-alkenes and iso- pentenyl esters of unsaturated fatty acids were the most attractive samples. Isopentenyl esters of unsaturated fatty acids were the key compounds in inducing male inspections as well as stimulating pounces and copulatory attempts. Virgin and nesting gynes differed clearly in the relative and absolute amounts of the volatiles on the cuticle. The total amount of volatiles was significantly higher in virgin gynes and decreased in breeding queens. Hydrocarbons were the dominant group of compounds in both groups of females. The relative amounts of the wax-type ester, hexadecyl oleate, iso- pentenyl esters, and a hitherto unidentified steroid were higher in attractive virgin gynes, while the relative proportions of hydrocarbons and lactones dominated in nesting queens. The site of sex pheromone production in attractive young L. (Evylaeus) malachurum gynes remains unknown. Head glands or Dufour's gland secretions may be involved. Another possible source of the `active principle' found among the cuticular lipids could be glandular cells of the epidermis. The significance of modulation of female sex pheromone composition is discussed in terms of a reduction in mating expenditures. Received: 30 April 1998 / Accepted after revision: 24 July 1998