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Carbonic anhydrase activity and biomineralization process in embryos, larvae and adult blue mussels Mytilus edulis L
Abstract
To decide whether a physiological role can be attributed to enzymatic activity with respect to crystal formation and biomineralization
of the first larval shell, carbonic anhydrase (CA) activity was measured in embryos and larvae of the blue mussels Mytilus edulis L. Also, CA activity was determined in the mantle edge and gonads of adult mussels with different shell length and condition
index. The intention was to find a possible correlation between CA activity and adult shell calcification, i.e. gonadal maturation.
The comparison of CA activity in different developmental stages of mussels and the results of an X-ray diffraction study of
biomineralization processes in embryonic and larval shells indicate that CA activity is maximal at the end of several developmental
stages. Consequently, the increase in CA activity precedes some physiological changes, i.e. the somatoblast 2d formation and
the occurrence of the first calcite and quartz crystals in embryos, shell field formation in the gastrula stage, shell gland
and periostracum production in trochophores, and rapid aragonite deposition in larval prodissoconch I and prodissoconch II
shells. Furthermore, it was found that in adult mussels CA activity was quite variable and that in the mantle edge it was
frequently inversely related to the activity in the gonad.
... Regulation of pHi can also be achieved via intracellular modulation of bicarbonate concentrations via carbonic anhydrases and a range of other proton equivalent exchange processes. Carbonic anhydrases can produce HCO 3 − from intracellular CO 2 and their role in biomineralisation has been studied in a range of molluscs (Medakovic 2000;Marie et al. 2008). However, there is limited information regarding the role of carbonic anhydrases in pH i regulation, particularly in calcifying tissue. ...
... In contrast to the results obtained on the role of extracellular HCO 3 − in C. gigas pH i regulation in mantle cells, significantly lower pH i recovery rates in the presence of the carbonic anhydrase inhibitor, acetazolamide were observed. The enzyme carbonic anhydrase facilitates the reversible hydration of CO 2 to HCO 3 − and has long been suggested to be an important enzyme in mollusc shell forming tissue such as mantle epithelia (Medakovic 2000;Miyamoto et al. 2005;Yu et al. 2006;Aguilera et al. 2017). Seventeen genes encoding both extracellular and cytosolic isoforms of carbonic anhydrase proteins have been identified in the C. gigas genome (Zhang et al. 2012). ...
... Our results demonstrate that the activity of specific ion regulatory proteins such as NHEs and carbonic anhydrase are crucial for acid-base regulation. Interestingly, these proteins have also been associated with biomineralization (Medakovic et al. 2000;Zhang et al. 2012). Specifically, a suite of novel molecular studies lend support to the role of these carbonic anhydrases in acquisition of inorganic carbon during calcification (Wang et al. 2017;Koh et al. 2018;Chew et al. 2019). ...
Shell formation and repair occurs under the control of mantle epithelial cells in bivalve molluscs. However, limited information is available on the precise acid–base regulatory machinery present within these cells, which are fundamental to calcification. Here, we isolate mantle epithelial cells from the Pacific oyster, Crassostrea gigas and utilise live cell imaging in combination with the fluorescent dye, BCECF-AM to study intracellular pH (pHi) regulation. To elucidate the involvement of various ion transport mechanisms, modified seawater solutions (low sodium, low bicarbonate) and specific inhibitors for acid–base proteins were used. Diminished pH recovery in the absence of Na+ and under inhibition of sodium/hydrogen exchangers (NHEs) implicate the involvement of a sodium dependent cellular proton extrusion mechanism. In addition, pH recovery was reduced under inhibition of carbonic anhydrases. These data provide the foundation for a better understanding of acid–base regulation underlying the physiology of calcification in bivalves.
... A reference map is typically developed from TEFs of sedentary adults collected from potential source populations (DiBacco and Levin, 2000). However, larval and adult mussels precipitate different calcium carbonate polymorphs during shell growth, which might influence TEFs (Medaković, 2000;Weiss et al., 2002;Dalbeck et al., 2006;Strasser et al., 2008). The larval shell (prodissoconch) of Mytilus edulis first appears toward the end of gastrulation (24-48 h. ...
... The concentration of several trace elements differed between larval and juvenile mussel shells reared in either controlled laboratory conditions or in situ chambers at several field sites in the northern Gulf of Maine (Figs. 2, 4). There are likely several interacting factors influencing structures and processes of biomineralization, such as differences in the crystal structure of CaCO 3 polymorphs (de Leeuw and Parker, 1998;Medaković, 2000;Dorval et al., 2005;Dalbeck et al., 2006), ontogenic shifts in ion transport pathways (Weiner and Dove, 2003) and the relative frequency of element insertion within aragonitic, calcitic and proteinaceous shell structures (Lorens and Bender, 1980;Addadi et al., 2003;Schöne et al., 2010;Littlewood et al., 2017;Dauphin et al., 2018). ...
... Understanding differences in likely transport mechanisms (Weiner and Dove, 2003) and trace element incorporation within unique calcium carbonate polymorphs crystallized and phase transformed across successive ontogenetic stages (Furuhasi et al., 2009) is key to explaining why TEFs vary spatially and temporally. Intense enzymatic activity occurs at the site of crystallization, contributing to ontogenetic changes in calcium carbonate polymorph, size and crystal shape (Medaković, 2000;Marin and Luquet, 2004;Barthelat et al., 2009;Cartwright et al., 2009). Larval aragonitic shell content increases over time from amorphous calcium carbonate (ACC; Medaković, 2000), while newly settled dissoconch and juvenile shells are composed of surface trilayered periostracum (Harper, 1997) underlain by prismatic calcite and nacreous aragonite (Marin and Luquet, 2004). ...
The ability to accurately estimate population connectivity and larval dispersal among mussel populations in the Gulf of Maine is important to better understand ongoing declines in blue mussel abundances. Such efforts are crucial for crafting conservation strategies targeting important spawning and settlement sites necessary to support threatened local shellfisheries, and to mitigate the potential effects of rapid climate change on larval dispersal and survivorship. Trace element fingerprints can be used to infer larval dispersal and population connectivity, but require a reference map of geographic variation in elemental fingerprints to infer natal sites of settled mussels. Previous work has suggested rearing mussel larvae in situ to create a reference map because biomineralization differs between larval and post-metamorphic mussels, which might lead to differences in trace element fingerprints. To test whether elemental fingerprints differed between larval and juvenile (post-metamorphic) mussels (Mytilus edulis), we reared them in situ and under controlled laboratory conditions. Trace element concentrations in larval and juvenile shell matrices were quantified using laser ablation inductively coupled plasma mass spectrometry (LA-ICPMS). The majority of trace element concentrations, normalized to calcium, differed between larval and juvenile mussels, resulting in elemental fingerprints that were consistently distinct between age classes. Larval and juvenile fingerprints differed consistently whether they were reared in the lab or field, probably reflecting age-specific differences in biomineralization. To use trace element fingerprints to estimate larval dispersal and population connectivity, our results suggest that a spatial map of elemental fingerprints should be created by rearing larval, not juvenile, mussels at potential source populations. Our results are consistent with what has been found for other mussels and thus may be true for all mussels, and likely true for any bivalves with age-specific differences in biomineralization.
... Carbonic anhydrase is a key enzyme responsible for increasing the localized concentration of CO 3 2at key locations in the bodies of molluscs where shell formation occurs (Lindskog, 1997;Marin et al., 2008;Zhang and Zhang, 2006). For example, high activities of carbonic anhydrase were observed immediately prior to shell formation during the development of blue mussel (Mytilus edulis) larvae (Medakovic, 2000). The function of carbonic anhydrase is affected by the presence of heavy metal ions. ...
... We observed zinc concentrations of only 5 -9 ppb in the seawater in this experiment ( Fig. 2A & B), yet EDTA still significantly reduced the accumulation of zinc by P. canaliculus larvae (Fig. 1, Table 1). Zinc is an essential element, required for the function of carbonic anhydrase for example (Medakovic, 2000), so reduction of its bioavailability may not be beneficial. Results from this study suggest that adding EDTA could be altering the distribution of zinc (Fig. 3), as well as reducing overall zinc concentrations within shellfish larvae. ...
... Interestingly, zinc was concentrated around the margins of shells in larvae developed without EDTA, but in larvae raised in seawater with EDTA, zinc was more evenly distributed throughout the larva (Fig. 3). Zinc coordinates with the enzyme carbonic anhydrase, which is an important enzyme involved in bio-mineralisation of the CaCO 3 shell (Medakovic, 2000;Miyamoto et al., 1996;Zhang and Zhang, 2006). This could explain the observed localisation of zinc in areas of high shell formation. ...
Heavy metal pollution is a concern in many coastal locations where it is frequently deleterious to the survival of young shellfish. Consequently, a great number of commercial shellfish hatcheries around the world rely on the addition of ethylenediaminetetraacetic acid (EDTA) to seawater to ensure successful larval production. Despite the importance of this practice to global shellfish production the mode of action of EDTA in larval production remains undetermined. It is assumed EDTA chelates heavy metals in seawater preventing interference in larval development. Larval mussels (Perna canaliculus) raised in seawater with 3 μM EDTA had a 15 fold higher yield than those without EDTA. The concentration and spatial arrangement of heavy metals in larvae as determined by Inductively Coupled Plasma Mass Spectrometry (ICPMS) and X-ray Fluorescence Microscopy (XFM) was consistent with reduced bioavailability of several metals, especially copper and zinc. This is the first study to confirm the effectiveness of EDTA for managing metal pollution commonly encountered in coastal shellfish hatcheries.
... Furthermore, in bivalves, the process of biological mineralization is closely related to shell growth. Carbonic anhydrase is one of the most important indicators of biological mineralization (Medaković, 2000;Cardoso et al., 2019). ...
... Studies on blue mussels (M. edulis) have confirmed that carbonic anhydrase activity is essential for rapid shell development (Medaković, 2000). Wilbur and Anderson (1950) also reported that enzyme content might increase with the development of shellfish. ...
Pearl oyster (Pinctada fucata martensii) is the main species cultured for marine pearls in the world. A breeding program was carried out for desirable production traits, including high growth rate, and a fast-growing selective strain of pearl oysters was established. In the current study, we compared the growth characteristics between a selective strain and a cultured population of P. f. martensii in Beihai, Guangxi Province, China. Large size (SL) and small size (SS) individuals of the selective strain were selected, and the differences of physiological and metabolic indexes, such as feeding, respiration, excretion, and enzyme activities between SL and SS and cultured population (CL), were also compared. The results showed that at the age of 6 months, pearl oysters of the selective strain were 14.61% larger than CL, and the proportion of SL (30–40 mm) was 59%, which was two times higher than CL (28%). SL with a rapid growth rate had a high clearance rate (CR), and the CR of SL was about 1.8 times higher than that of CL and 5 times higher than that of SS. In addition, the activities of digestive enzymes (amylase, pepsin, and lipase) and growth-related carbonic anhydrase enzymes in SL were higher than those in the other two groups (p < 0.05). SS with a slow growth rate had higher oxygen consumption (OCR) and ammonia excretion (AER) rates than SL and CL (p < 0.05). Our results suggest that the rapid growth of the selective strain P. f. martensii can be attributed to increased energy intake and reduced energy consumption.
... In addition to Ca, a variety of metals e.g. Zn (Medaković, 2000) and Mg (Jacob et al., 2008), as well as trace amounts of Sr, Mn, B, Co, Ti, Ba, Li appear important for bivalve shell formation, and environmental factors (e.g. pH and metal bioavailability) significantly influence Greenshell™ mussel shell composition (Norrie et al., 2019(Norrie et al., , 2018. ...
... The relative decrease in fitness after D2 suggests that EDTA may be binding elements essential for larval growth, such as Zn (McDougall et al., 2019(McDougall et al., , 2020. Because Zn is essential for shell mineralisation (Medaković, 2000); decreased bioavailability of Zn for extended periods of time in the EDTA treatment may result in less Zn uptake for the larvae to perform necessary biological functions. Conversely, while Zn is essential, excess concentrations can also be detrimental to developing bivalve larvae (Brereton et al., 1973;Watling, 1982), as discussed below. ...
The use of ethylenediaminetetraacetic acid (EDTA) during bivalve hatchery production is thought to improve larval yields due to the reduced exposure to toxic metals (such as Cu); however, few studies have focused on the bioavailability of metals during the rearing process. Greenshell™ mussels (Perna canaliculus) were reared for 48 h with and without EDTA (12 µM) exposure and larvae were subsequently raised to 21 days post-fertilisation with and without EDTA exposure. Survival, shell length, algal ingestion rate, swimming activity, total metal concentration in water, bioavailable metal concentrations and larval metal accumulation were monitored for the 21 day period. Larval fitness (specifically D-yields) was improved on day 2 in the EDTA treatment, whereas an overall negative effect of EDTA treatment on fitness was observed on day 10 and 21. During the first 48 h, increased survival in the EDTA treatment is believed to be due to the reduction of bioavailable Zn concentrations in the rearing seawater. No other metal (essential or non-essential) displayed a consistent trend when comparing metal bioavailability to any of the fitness parameters measured throughout the experiment. Though the measured metal bioavailability was not clearly linked to fitness, the uptake of Al, P, Cr, Fe, Co, Ni, Zn, As, Cd, and Hg by P. canaliculus was reduced during the first 48 h, suggesting that the biological regulation of these elements is just as important as the bioavailability. Overall, treatment of the rearing seawater with 12 µM EDTA is effective for improving Greenshell™ mussel larval yields by decreasing metal bioavailability during the first two days of development but has minimal benefit between day 2 and 21.
... The molecular components involved in shell formation have been largely investigated in adults and larval stages of different bivalves (oysters, clams, mussels) [9][10][11][12][13][14][15][16][17][18][19][20][21][22][23]. Transcriptomics and proteomics data have identified several genes that play important roles in the biomineralization process, as well as a number of shell matrix proteins (SMPs). ...
... However, the role of each component in the transition from the trocophora to the first shelled embryo, when the blueprint for calcification is first established, is not fully understood. In particular, despite data being available on shell calcification in early larval stages [20][21][22][23], and also in relation to ocean acidification [24,25], much less is known about the ontogeny of the organic matrix before calcification occurs [2]. Former studies underlined the role of chitin deposition [5][6][7]. ...
Bivalve biomineralization is a highly complex and organized process, involving several molecular components identified in adults and larval stages. However, information is still scarce on the ontogeny of the organic matrix before calcification occurs. In this work, first shell formation was investigated in the mussel Mytilus galloprovincialis. The time course of organic matrix and CaCO3 deposition were followed at close times post fertilization (24, 26, 29, 32, 48 h) by calcofluor and calcein staining, respectively. Both components showed an exponential trend in growth, with a delay between organic matrix and CaCO3 deposition. mRNA levels of genes involved in matrix deposition (chitin synthase; tyrosinase- TYR) and calcification (carbonic anhydrase; extrapallial protein) were quantified by qPCR at 24 and 48 hours post fertilization (hpf) with respect to eggs. All transcripts were upregulated across early development, with TYR showing highest mRNA levels from 24 hpf. TYR transcripts were closely associated with matrix deposition as shown by in situ hybridization. The involvement of tyrosinase activity was supported by data obtained with the enzyme inhibitor N-phenylthiourea. Our results underline the pivotal role of shell matrix in driving first CaCO3 deposition and the importance of tyrosinase in the formation of the first shell in M. galloprovincialis.
... Using crossed polarized microscopy we could demonstrate that the growing shell is birefringent from 22 hpf onwards ( Fig. 1i, j, Supplementary Movie 1), indicating the presence of a high fraction of crystalline aragonite. Presence of aragonite in larval M. edulis shells from 22 hpf (at 18°C) onwards has previously been observed using XRD 21 and early veliger shells of other bivalve species also consist of aragonite 22,23 . Video recordings of swimming and rotating larvae under crossed polarized light indicate that the entire shell is birefringent (Supplementary Movie 1). ...
... The mechanisms underlying this pH regulatory effort are yet unexplored in bivalves. Rapid aragonite deposition in mussel larvae has been correlated with increased activity of carbonic anhydrase, but it is unclear if it plays a role in CS carbonate chemistry regulation 21 . However, based on larval oyster gene expression data, all typical pH regulatory proteins that are commonly utilized to control extracellular and intracellular pH in marine metazoans are expressed in bivalve larvae 28 . ...
Understanding mollusk calcification sensitivity to ocean acidification (OA) requires a better knowledge of calcification mechanisms. Especially in rapidly calcifying larval stages, mechanisms of shell formation are largely unexplored—yet these are the most vulnerable life stages. Here we find rapid generation of crystalline shell material in mussel larvae. We find no evidence for intracellular CaCO3 formation, indicating that mineral formation could be constrained to the calcifying space beneath the shell. Using microelectrodes we show that larvae can increase pH and [CO3²⁻] beneath the growing shell, leading to a ~1.5-fold elevation in calcium carbonate saturation state (Ωarag). Larvae exposed to OA exhibit a drop in pH, [CO3²⁻] and Ωarag at the site of calcification, which correlates with decreased shell growth, and, eventually, shell dissolution. Our findings help explain why bivalve larvae can form shells under moderate acidification scenarios and provide a direct link between ocean carbonate chemistry and larval calcification rate.
... In plants, algae and some bacteria, α-CAs assume a vital role in the photosynthesis process and biosynthesis reaction (Moroney et al., 2001). α-CAs accelerate the interconversion between CO 2 and HCO 3 and play a vital role during the calcium carbonate (CaCO 3 ) formation in mollusks, such as acid-base balance, transport of CO 2 /HCO 3 − , calcification and biomineralization (Medakovic, 2000). Nacrein, the first molluscan matrix protein to be identified, is involved in the formation of the prismatic and nacreous layers (Miyamoto and Matsushiro, 1996;Voigt et al., 2014;Miyamoto et al., 2005). ...
... Western blot detected that HcCA3 protein existed in F, which may be related to the rich features of CA. In general, CAs occur in different organizations of the same organism and undertake different physiological functions: ion transport; acid-base balance; carbon dioxide transport; biosynthesis (Medakovic, 2000;Li, 2013;Sharma and Bhattacharya, 2010). Foot directly is exposed in the water when moving. ...
... Carbonic anhydrase plays key roles in oyster shell mineralization (Parker et al., 2010) and skeleton deposition in scleractinian corals (Hofmann et al., 2012) through the interconversion of bicarbonate and carbon dioxide. Although up-regulation of CA could accelerate bicarbonate formation and help in maintaining several physiological functions, such as respiration, ion transport, and acid-base regulation, increasing CA activity generally led to increased calcification in larval oysters (Medakovi c, 2000). One possible explanation for the increase in CA activity observed in our study is that the Pacific oyster was able to adjust the pH at the site of calcium carbonate deposition (Gazeau et al., 2010;Hofmann & Todgham, 2010;Parker et al., 2013), even though several of the proteins involved in calcification were down-regulated. ...
... This would explain why the larvae in the current study were able to survive, calcify, and grow at pH 7.4, albeit at a slower rate than in the control treatment. There is growing evidence to support this hypothesis in many calcifying marine species (Medakovi c, 2000;Fabry et al., 2008;Hofmann et al., 2012). ...
The metamorphosis of planktonic larvae of the Pacific oyster (Crassostrea gigas) underpins their complex life-history strategy by switching on the molecular machinery required for sessile life and building calcite shells. Metamorphosis becomes a survival bottleneck, which will be pressured by different anthropogenically induced climate change-related variables. Therefore, it is important to understand how metamorphosing larvae interact with emerging climate change stressors. To predict how larvae might be affected in a future ocean, we examined changes in the proteome of metamorphosing larvae under multiple stressors: decreased pH (pH 7.4), increased temperature (30 °C), and reduced salinity (15 psu). Quantitative protein expression profiling using iTRAQ-LC-MS/MS identified more than 1300 proteins. Decreased pH had a negative effect on metamorphosis by down-regulating several proteins involved in energy production, metabolism, and protein synthesis. However, warming switched on these down-regulated pathways at pH 7.4. Under multiple stressors, cell signaling, energy production, growth, and developmental pathways were up-regulated, although metamorphosis was still reduced. Despite the lack of lethal effects, significant physiological responses to both individual and interacting climate change related stressors were observed at proteome level. The metamorphosing larvae of the C. gigas population in the Yellow Sea appear to have adequate phenotypic plasticity at the proteome level to survive in future coastal oceans, but with developmental and physiological costs.
... A notable exception to this is Yokoo's et al. [20] comprehensive investigation, detailing the biomineralization characterictics of Pinctada fucata ontogeny. Additional piecemeal descriptions of these shell characteristics for larvae are known for a variety of taxa including: gastropods [21][22][23][24], mussel [25][26][27], edible oysters [28,29], pearl oyster [30] and clams [17]. Yokoo's et al. [20] report in combination with the aforementioned studies suggest a general trend for molluscan larval shell mineralization. ...
... In some cases this outer prismatic layer is very thin or totally absent [31]. Most recently, several studies have noted a generic predominance of amorphous calcium carbonate (ACC) in the initial deposited mineral phase of the prodissoconch I along with poorly crystalline aragonite [18,27,32,33]. These investigations have attributed ACC as an important precursor to molluscan larval shell formation and potentially adult mineralization. ...
Molluscan larval ontogeny is a highly conserved process comprising three principal developmental stages; trochophore, veliger and metamorphosis into the juvenile. A characteristic that is unique to each of these stages is shell design, termed prodissoconch I, prodissoconch II and dissoconch in bivalves. These shells vary in morphology, mineralogy and microstructure. The discrete temporal transitions in shell biomineralization between these larval stages are utilized in this study to investigate transcriptional involvement in several distinct biomineralization events. Scanning electron microscopy and X-ray diffraction analysis of P. maxima larvae and juveniles collected throughout post-embryonic ontogenesis, document the mineralogy and microstructure of each shelled stage as well as establishing a timeline for transitions in biomineralization. P. maxima larval samples most representative of these biomineralization distinctions and transitions were analyzed for differential gene expression with the microarray platform PmaxArray 1.0. A number of known shell matrix genes and novel transcripts are reported as differentially expressed in correlation to the mineralization events of P. maxima larval ontogeny. However, only a single transcript, PM066, was noted as being expressed before and after the transition to the adult shell design. No other known/putative adult shell matrix genes from P. maxima were detected in association with the larval shells prodissococh I and II suggesting that there expression is either below detection limits or an almost entirely different sets of genes are potentially responsible for larval and adult shell mineralization. This interdisciplinary investigation has linked the shell developments of P. maxima larval ontogeny with corresponding gene expression profiles, furthering the elucidation of bivalve development and shell biomineralization.
... At metamorphosis, the free-swimming larvae of serpulid worms undergo a pelagic-benthic transformation when they attach to suitable substrates and actively produce CaCO 3. The calcium secreting glands control the calcification process (Bubel, 1983;Hedley, 1956a,b), using HCO 3 À and Ca 2+ as the calcification reactants (Roleda et al., 2012). The early calcification products commonly involve complex succession, for example, formation of the larval spicules in sea urchins and the early larval shells in mussels, reorganized from highly the disordered amorphous calcium carbonate (ACC) phase into mature crystalline minerals (Beniash et al., 1997;Medaković , 2000;Addadi et al., 2003;Politi et al., 2008). The structure of the ACC lacks any long-range order, but commonly contains short-range order of the designated mineral product, i.e. calcite or aragonite. ...
... A pattern of biomineral succession over development has been reported in a few groups of marine invertebrates. For example, the urchin and mollusk larvae are known to have an amorphous precursor prior to the formation of calcite and aragonite mineral (Beniash et al., 1997;Medaković , 2000). Larval oysters, for example, produce aragonitic shells and switch to producing calcite after metamorphosis in the sessile juvenile stage (Medaković et al., 1997). ...
The serpulid tubeworm, Hydroides elegans, is an ecologically and economically important species whose biology has been fairly well studied, especially in the context of larval development and settlement on man-made objects (biofouling). Nevertheless, ontogenetic changes associated with calcareous tube composition and structures have not yet been studied. Here, the ultrastructure and composition of the calcareous tubes built by H. elegans was examined in the three early calcifying juvenile stages and in the adult using XRD, FTIR, ICP-OES, SEM and Raman spectroscopy. Ontogenetic shifts in carbonate mineralogy were observed, for example, juvenile tubes contained more amorphous calcium carbonate and were predominantly aragonitic whereas adult tubes were bimineralic with considerably more calcite. The mineral composition gradually shifted during the tube development as shown by a decrease in Sr/Ca and an increase of Mg/Ca ratios with the tubeworm's age. The inner tube layer contained calcite, whereas the outer layer contained aragonite. Similarly, the tube complexity in terms of ultrastructure was associated with development. The sequential appearance of unoriented ultrastructures followed by oriented ultrastructures may reflect the evolutionary history of serpulid tube biominerals. As aragonitic structures are more susceptible to dissolution under ocean acidification (OA) conditions but are more difficult to be removed by anti-fouling treatments, the early developmental stages of the tubeworms may be vulnerable to OA but act as the important target for biofouling control.
Copyright © 2015. Published by Elsevier Inc.
... form a family of enzymes that catalyze the process of reversible hydration of CO 2 to yield HCO 3 − and H + in the carbonic acid equilibrium (CO 2 + H 2 O ↔ HCO 3 − + H + ) (Badger and Price, 1994). CAs can participate in various physiological processes, such as respiration, pH homeostasis, ion transport, photosynthesis, synthesis of fatty acid and amino acid and biomineralization (Henry, 1996;Medakovic, 2000). To date, five CA (α, β, γ, δ, and ζ-CAs) families have been identified, and the α-CA is the predominant group in metazoans (Bertucci et al., 2013). ...
... Recent studies reveal that mollusks express several CA isoforms in the mantle (Le Roy et al., 2012;Werner et al., 2013;Zhang et al., 2012). The existence of different CAs in the mantle results in versatile functional roles of CAs, including acid-base regulation, ion transport, respiration and biomineralization (Medakovic, 2000). In the present study, the high expression level of HcCA was found in the mantle center and the mantle pallial of adult mussel, suggesting that HcCA might participate in the mantle metabolic functions. ...
... Carbonic anhydrase activity. CA activity was determined for the mantle tissue and extrapallial fluid from the posterior abductor muscle 23 . CA has already been observed in the acid-soluble matrices (ASMs) of the nacreous layer of mussel shells 24 and in mussel soft tissue 23 thus these areas were targeted. ...
... CA has already been observed in the acid-soluble matrices (ASMs) of the nacreous layer of mussel shells 24 and in mussel soft tissue 23 thus these areas were targeted. Mantle tissue and extrapallial fluid CA activity was determined by the colourimetric micromethod of analysis 23,25,26 . The activity units or enzyme units (EU) were calculated by the equation EU 5 (To-T)/T, where T and To are the reaction times for the pH change with and without the catalyst respectively 23 . ...
Ocean acidification is altering the oceanic carbonate saturation state and threatening the survival of marine calcifying organisms. Production of their calcium carbonate exoskeletons is dependent not only on the environmental seawater carbonate chemistry but also the ability to produce biominerals through proteins. We present shell growth and structural responses by the economically important marine calcifier Mytilus edulis to ocean acidification scenarios (380, 550, 750, 1000 µatm pCO2). After six months of incubation at 750 µatm pCO2, reduced carbonic anhydrase protein activity and shell growth occurs in M. edulis. Beyond that, at 1000 µatm pCO2, biomineralisation continued but with compensated metabolism of proteins and increased calcite growth. Mussel growth occurs at a cost to the structural integrity of the shell due to structural disorientation of calcite crystals. This loss of structural integrity could impact mussel shell strength and reduce protection from predators and changing environments.
... form a family of enzymes that catalyze the process of reversible hydration of CO 2 to yield HCO 3 − and H + in the carbonic acid equilibrium (CO 2 + H 2 O ↔ HCO 3 − + H + ) (Badger and Price, 1994). CAs can participate in various physiological processes, such as respiration, pH homeostasis, ion transport, photosynthesis, synthesis of fatty acid and amino acid and biomineralization (Henry, 1996;Medakovic, 2000). To date, five CA (α, β, γ, δ, and ζ-CAs) families have been identified, and the α-CA is the predominant group in metazoans (Bertucci et al., 2013). ...
... Recent studies reveal that mollusks express several CA isoforms in the mantle (Le Roy et al., 2012;Werner et al., 2013;Zhang et al., 2012). The existence of different CAs in the mantle results in versatile functional roles of CAs, including acid-base regulation, ion transport, respiration and biomineralization (Medakovic, 2000). In the present study, the high expression level of HcCA was found in the mantle center and the mantle pallial of adult mussel, suggesting that HcCA might participate in the mantle metabolic functions. ...
... Higher levels of CA2 2 −ΔΔCt values were found in male groups with respect to females, and possibly associated with metabolic profiles, hormonal state or fitness strategies (Ji et al. 2013;Mikulski et al. 2011;Wong et al. 2014). It was reported that CA activity was found at different levels during the life cycle of M. edulis, being highest at the end of the developmental stages (Medaković 2000). Interestingly, in Wang et al. (2017), expression of the CAII-1 gene in Crassostrea gigas exposed to low pH was downregulated only in male gonads, in contrast with a significant upregulation in other non-reproductive tissue samples. ...
Plastic pollution and changes in oceanic pH are both pressing environmental issues. Little emphasis, however, has been placed on the influence of sex and gametogenesis stage when investigating the effects of such stressors. Here, we examined histology and molecular biomarkers of blue mussels Mytilus edulis exposed for 7 days to a pH 7.7 scenario (− 0.4 units) in combination with environmentally relevant concentrations (0, 0.5 and 50 µg/L) of the endocrine disrupting plasticiser di-2-ethylhexyl phthalate (DEHP). Through a factorial design, we investigated the gametogenesis cycle and sex-related expression of genes involved in pH homeostasis, stress response and oestrogen receptor-like pathways after the exposure to the two environmental stressors. As expected, we found sex-related differences in the proportion of developing, mature and spawning gonads in histological sections. Male gonads also showed higher levels of the acid-base regulator CA2, but females had a higher expression of stress response-related genes (i.e. sod, cat, hsp70). We found a significant effect of DEHP on stress response-related gene expression that was dependent on the gametogenesis stage, but there was only a trend towards downregulation of CA2 in response to pH 7.7. In addition, differences in gene expression between males and females were most pronounced in experimental conditions containing DEHP and/or acidified pH but never the control, indicating that it is important to consider sex and gametogenesis stage when studying the response of mussels to diverse stressors.
... Solid state NMR analysis of phosphate species in abalone larvae. H tuberculata is a marine gastropod of economic interest and a model for studying basic mechanisms of mollusc shell formation which is composed of aragonite and a variable amount of ACC at the early stages of development 37 similarly to other molluscs 38,39 . Three freshly collected larval stages (48, 72, and 96 h post fertilization (hpf), Fig. 1a) were analyzed by solid state NMR without any chemical or thermic treatment to probe larvae in their native hydration state. ...
The presence of phosphate from different origins (inorganic, bioorganic) is found more and more in calcium carbonate-based biominerals. Phosphate is often described as being responsible for the stabilization of the transient amorphous calcium carbonate phase. In order to specify the composition of the mineral phase deposited at the onset of carbonated shell formation, the present study investigates, down to the nanoscale, the growing shell from the European abalone Haliotis tuberculata, using a combination of solid state nuclear magnetic resonance, scanning transmission electron microscope and spatially-resolved electron energy loss spectroscopy techniques. We show the co-occurrence of inorganic phosphate with calcium and carbonate throughout the early stages of abalone shell formation. One possible hypothesis is that this first-formed mixed mineral phase represents the vestige of a shared ancestral mineral precursor that appeared early during Evolution. In addition, our findings strengthen the idea that the final crystalline phase (calcium carbonate or phosphate) depends strongly on the nature of the mineral-associated proteins in vivo. Phosphate involvement in calcium carbonate biominerals raises questions on biomineralisation pathways. Here, the authors explore the presence of phosphate in the growing shell of the European abalone and suggest a shared mixed mineral ancestral precursor with final crystal phase being selected by mineral-associated proteins.
... The activity of this enzyme was found to increase prior to important physiological changes, e.g. the shell field formation in the gastrula stage of Mytilus edulis larvae. Furthermore, carbonic anhydrase activity increased before rapid aragonite deposition in larval prodissoconch I and II shells of the blue mussel (MEDAKOVIĆ 2000). Although carbonic anhydrase is a fairly common enzyme involved in many physiological processes, it may well play a role in mollusc calcification processes. ...
The emissions of carbon dioxide into the atmosphere have risen strongly since the
beginning of the industrialisation. About 48% of the anthropogenic CO2 emissions are
taken up by the world’s oceans. The excess carbon dioxide interferes with the ocean’s
capacity to buffer changes in pH, the carbonate system, and causes the acidification of the
seawater. A decrease in the pH value of the ocean has negative consequences for calcifying
metazoans, e.g. bivalves, because their calcification process may be negatively influenced.
Larval stages are more sensitive to declining pH values than adults, but still there are only
few studies on larvae.
In the present study, the effects of acidified seawater on growth, survival, and shell
calcium content of larvae of the European oyster (Ostrea edulis L.) were investigated. The
seawater was acidified by addition of HCl, and five pH values below the recent pH were
created, decreasing in steps of 0.25 units. One treatment was left at the natural condition,
another treatment was alkalised to simulate a pre-industrial pH value. The experiment was
performed in 2 liter glass bottles with separate aeration for each treatment. Air from the
headspaces of the bottles was pumped into the water, thus a mixing with outside air was
prevented.
Acidified seawater had a significantly negative effect on larval shell length. The smallest
shells and slowest growth were found in larvae cultivated at the lowest pH value. Survival
rates were not significantly affected by the pH value, although larvae in the alkalised
treatment survived seven days longer than in all the other treatments. An effect of the pH
value on the calcium content as an indicator of shell calcification could not be proven,
although this may be due to the processing of the samples.
... This process may be catalyzed by an enzymatic conversion by carbonic anhydrase (CA), which is present in many prokaryotes and virtually all eukaryotes (Hewett-Emmett and Tashian, 1996;Lionetto et al., 2016). This enzyme is essential in calcification in many organisms, including corals, sponges and coccolithophores (Bertucci et al., 2013;Medaković, 2000;Müller et al., 2013;Le Roy et al., 2014;Wang et al., 2017). Also for foraminiferal calcification it has been hypothesized that CA is used to enhance inorganic-carbon uptake. ...
Marine calcification is an important component of the global carbon cycle.
The mechanism by which some organisms take up inorganic carbon for the
production of their shells or skeletons, however, remains only partly known.
Although foraminifera are responsible for a large part of the global calcium
carbonate production, the process by which they concentrate inorganic carbon
is debated. Some evidence suggests that seawater is taken up by
vacuolization and participates relatively unaltered in the process of
calcification, whereas other results suggest the involvement of
transmembrane transport and the activity of enzymes like carbonic anhydrase.
Here, we tested whether inorganic-carbon uptake relies on the activity of
carbonic anhydrase using incubation experiments with the perforate, large
benthic, symbiont-bearing foraminifer Amphistegina lessonii. Calcification rates, determined by
the alkalinity anomaly method, showed that inhibition of carbonic anhydrase
by acetazolamide (AZ) stopped most of the calcification process. Inhibition
of photosynthesis either by 3-(3,4-Dichlorophenyl)-1,1-dimethylurea (DCMU)
or by incubating the foraminifera in the dark also decreased calcification
rates but to a lesser degree than with AZ. Results from this study show
that carbonic anhydrase plays a key role in biomineralization of
Amphistegina lessonii and indicates that calcification of those perforate, large benthic
foraminifera might, to a certain extent, benefit from the extra dissolved inorganic carbon (DIC), which
causes ocean acidification.
... Unlike the initial days of the experiment, gene expression at day 6 decreased at pH 8.0, and increased at pH 7.5, but a low expression level in both groups was observed on day 7. This expression pattern for CA agrees with studies in embryos and larvae of M. edulis with expression peaks of this enzyme observed at the interphase periods from one stage to the next (Medakovic, 2000). ...
Ocean acidification generates a decrease in calcium carbonate availability essential for biomineralization in organisms such as mollusks. This effect was evaluated on Panopea globosa exposing for 7 days umbonate veliger larvae to two pH treatments: experimental (pH 7.5) and control (pH 8.0). Exposure to pH 7.5 affected growth, reducing larval shell length from 5.15–13.34% compared to the control group. This size reduction was confirmed with electron microscopy, also showing shell damage. The physiological response showed an increase in oxygen consumption in larvae exposed to low pH with a maximum difference of 1.57 nmol O2 h⁻¹ larvae⁻¹ at day 7. The gene expression analyses reported high expression values for nicotinamide adenine dinucleotide (NADH) dehydrogenase and Perlucin in larvae at pH 7.5, suggesting a higher energetic cost in this larval group to maintain homeostasis. In conclusion, this study showed that acidification affected development of P. globosa umbonate veliger larvae.
... L'épithélium responsable de la minéralisation, appelé le manteau, est situé sous la coquille. Le liquide extrapalléal remplit la cavité et contient tous les ions et les protéines nécessaires au processus de biominéralisation ; les premiers sont amenés de l'hémolymphe vers la cavité au travers des cellules du manteau à l'aide de transporteurs ioniques, les secondes sécrétées par le manteau (Marin and Luquet, 2004 (Weiss et al, 2002) et Mytilus edulis (Medakovic, 2000) et (Allemand et al, 2011)dans les spicules d'oursins Paracentrotus lividus (Beniash et al, 1997) et ...
... The first 48 h of larval development is when the mineralisation of the shell is initiated with the deposition of mostly amorphous calcium carbonate, rather than crystalline calcite or aragonite (Medakovic 2000;Weiss et al. 2002). It is possible that larvae at this stage of development have less physio-chemical control over the acquisition of Ca for the formation of amorphous calcium carbonate shell, with an inability to control alternative metal ion incorporation resulting in toxicity effects due to substitution with non-essential metals. ...
The chelating agent ethylenediaminetetraacetic acid (EDTA) is used throughout the world to improve the yield of early stage D-larvae during bivalve hatchery production. Adding EDTA (12 μM) to seawater significantly increases the survival of Greenshell™ mussel (Perna canaliculus) larvae during their first 48 h of development. However, whether there are benefits of continuing to use EDTA beyond this first stage of larval development remain unknown and were tested in this study. After rearing for 48 h in the presence of EDTA, P. canaliculus larvae were experimentally raised to 22-day post-fertilisation in seawater with and without 12 μM EDTA. The survival, shell length growth, algal ingestion rate, swimming activity and potential toxic metal accumulation by the larvae were compared over this period. There were minimal benefits from continuing addition of EDTA. However, significant changes in metal concentrations within the larvae were observed. Zinc, cadmium and mercury were detected at significantly lower concentrations in 22-day-old larvae reared with EDTA versus those without EDTA. Collectively, the results indicate that the use of EDTA is critical only during the first 48 h of larval development, during which time larval shell formation is initiated and appears highly vulnerable to interference by heavy metal ions.
... Por otro lado, fosfatasa alcalina está involucrada en la absorción de fosfato y calcio durante los procesos de biomineralización de la capa nacarada en moluscos bivalvos (Chen et al., 2005) y durante la etapa de fijación esta capa nacarada, no inicia aun su formación por lo que la expresión de este gen se espera a niveles muy bajos. En condiciones normales de pH (pH 8.1), se ha observado que incrementa la actividad durante el inicio de la formación de la concha en el ostión plano O. edulis (Medaković, 2000). Esta enzima presenta una mayor expresión en el manto de C. gigas y ayuda a mantener el equilibrio ácido-base en la cavidad paleal, jugando un papel clave en la biomineralización (Ivanina et al., 2017). ...
Efecto de la acidificación oceánica simulada en el desarrollo inicial y en la modulación de la comunidad bacteriana de Crassostrea sikamea (Amemiya, 1928) y Crassostrea gigas (Thunberth, 1793)
... Some studies have also found a correlation between CA activity and shell formation (Fitzer et al., 2014b;Medaković and Lucu., 1994), and enzyme activity increases linearly with shell formation (Medaković, 2000). Nonetheless, the mechanisms at play only conferred a small benefit of parental exposure. ...
Transgenerational effects of multiple stressors on marine organisms are emerging environmental themes. We thus experimentally tested for transgenerational effects of seawater acidification and hypoxia on the early development traits of the mussel Mytilus edulis. Fertilization rate, embryo deformity rate, and larval shell length were negatively impacted by acidification, while hypoxia had little effect except for increasing deformity rates under control pH conditions. Offspring from low pH/O2 parents were less negatively affected by low pH/O2 conditions than offspring from control parents; however, low pH/O2 conditions still negatively affected developmental traits in offspring from acclimated parents compared to control seawater conditions. Our results demonstrate that experimental seawater acidification and hypoxia can adversely affect early developmental traits of M. edulis and that parental exposure can only partially alleviate these impacts. If experimental observations hold true in nature, it is unlikely that parental exposure will confer larval tolerance to ocean acidification for M. edulis.
... As well as directly causing shell dissolution, a number of other biological processes responsible for larval shell calcification, such as matrix protein production, chitin synthesis and enzymatic control are influenced by changes in seawater pCO 2 (Weiss et al., 2013). For example, the activity of carbonic anhydrase, an enzyme that catalyzes the reversible hydration of CO 2 to HCO 3 and H + , reaches its maximum activity at the end of each developmental stage and has been correlated with larval shell biomineralization (Gaume et al., 2011;Medakovic, 2000). In the mussel M. edulis, six months of incubation at 750 μatm pCO 2 (pH 7.5) significantly reduced carbonic anhydrase activity within the mantle tissue, explaining shell growth reduction (Fitzer et al., 2014b). ...
Ocean acidification is a major global stressor that leads to substantial changes in seawater carbonate chemistry, with potentially significant consequences for calcifying organisms. Marine shelled mollusks are ecologically and economically important species providing essential ecosystem services and food sources for other species. Because they use calcium carbonate (CaCO3) to produce their shells, mollusks are among the most vulnerable invertebrates to ocean acidification, with early developmental stages being particularly sensitive to pH changes. This study investigated the effects of CO2-induced ocean acidification on larval development of the European abalone Haliotis tuberculata, a commercially important gastropod species. Abalone larvae were exposed to a range of reduced pHs (8.0, 7.7 and 7.6) over the course of their development cycle, from early-hatched tro-chophore to pre-metamorphic veliger. Biological responses were evaluated by measuring the survival rate, morphology and development, growth rate and shell calcification. Larval survival was significantly lower in acidified conditions than in control conditions. Similarly, larval size was consistently smaller under low pH conditions. Larval development was also affected, with evidence of a developmental delay and an increase in the proportion of malformed or unshelled larvae. In shelled larvae, the intensity of birefringence decreased under low pH conditions, suggesting a reduction in shell mineralization. Since these biological effects were observed for pH values expected by 2100, ocean acidification may have potentially negative consequences for larval recruitment and persistence of abalone populations in the near future.
... DCF did not significantly affect shell size and mineralization, this latter eval- uated by polarized light microscopy. When the mineral composition of the shell was analyzed by XRD, the results indicate that aragonite was dominant in control shells, while calcite was present in traces (≤1%), in line with those previously reported in mussel and oyster embryos at the same stage, with the presence of small amounts of calcite crystals that play a role in the further development of larval shells, acting as ara- gonite crystallization centres (Medakovic, 2000 and ref. quoted therein). Interestingly, in DFC-treated samples, the mineral phase was represented only by aragonite. ...
Diclofenac-DCF, one of the most widely prescribed non-steroidal anti-inflammatory drug, is globally detected in environmental compartments. Due to its occurrence in freshwater and potential impact on aquatic organisms, it has been added to the watch list of chemicals in the EU Water Directive; consequently, research on the impact of DCF in model aquatic organisms has great regulatory implications towards ecosystem health. DCF is also detected in coastal waters at concentrations from ng/L to 1 μg/L, as well as in marine organisms, such as the mussel Mytilus. Increasing evidence indicates that environmental concentrations of DCF have multiple impacts in adult mussels. Moreover, in M. galloprovincialis, DCF has been shown to affect early embryo development.
The developmental effects of DCF in mussels were further investigated. DFC (1 and 10 μg/L) was added at different times post-fertilization (30 min and 24 hpf) and the effects were compared in the 48 hpf embryotoxicity assay. Shell mineralization and morphology were investigated by polarized light microscopy, X-Ray Spectrometry-XRD and Scanning Electron Microscopy-SEM. Transcriptional profiles of 12 selected genes physiologically regulated across early embryo development were assessed at 24 and 48 hpf.
DCF induced shell malformations, irrespectively of concentration and time of exposure. DCF phenotypes were characterized by convex hinges, undulated edges, fractured shells. However, no changes in biomineralization were observed. DCF affected gene transcription at both times pf, in particular at 1 μg/L. The most affected genes were those involved in early shell formation (CS, CA, EP) and biotransformation (ABCB, GST). The results confirm that Mytilus early development represents a significant target for environmental concentrations of DCF. These data underline how the standard embryotoxicity assay, in combination with a structural and transcriptomic approach, represents a powerful tool for evaluating the early impact of pharmaceuticals on mussel embryos, and identification of the possible underlying mechanisms of action.
... The digestive gland showed a significantly higher specific CA activity with respect to other organs such as gills and mantle, where CA has been functionally related to CO 2 exchange with the environmental water or calcium carbonate deposition in the shell (Medakovic, 2000;Cudennec et al., 2006;Le Roy et al., 2012;Werner et al., 2013;Pavičić-Hamer et al., 2016). This result argues in favor of a relevant physiological role played by CA in the digestive gland. ...
Carbonic anhydrase (CA) is a ubiquitous metalloenzyme, whose functions in animals span from respiration to pH homeostasis, electrolyte transport, calcification, and biosynthetic reactions. CA is sensitive to trace metals in a number of species. In mussels, a previous study demonstrated CA activity and protein expression to be enhanced in digestive gland by cadmium exposure. The aim of the present work was to investigate the functional meaning, if any, of this response. To this end the study addressed the possible involvement of CA in the lysosomal system response of digestive gland cells to metal exposure. The in vivo exposure to acetazolamide, specific CA inhibitor, significantly inhibited the acidification of the lysosomal compartment in the digestive gland cells charged with the acidotropic probe LysoSensor Green D-189, demonstrating in vivo the physiological contribution of CA to the acidification of the lysosomes. Under CdCl2 exposure, CA activity significantly increased in parallel to the increase of the fluorescence of LysoSensor Green charged cells, which is in turn indicative of proliferation and/or increase in size of lysosomes. Acetazolamide exposure was able to completely inhibit the cadmium induced Lysosensor fluorescence increase in digestive gland cells. In conclusion, our results demonstrated the functional role of CA in the lysosomal acidification of Mytilus galloprovincialis digestive gland and its involvement in the lysosomal activation following cadmium exposure. CA induction could physiologically respond to a prolonged increased requirement of H+ for supporting lysosomal acidification during lysosomal activation.
... Several of the differentially expressed transcripts are from genes known to code for parts of the shell matrix, such as nacrein [42], papilin (also a metalloprotease inhibitor; [40]), chitin-binding protein [43] and a protein with a beta-lactamase domain that is known to be part of the shell matrix, but with a currently unknown function [44]. Nacrein has a carbonic anhydrase domain [42], and has previously been shown to be strongly expressed prior to the initiation of shell formation in blue mussels [45]. Furthermore, several calcium binding proteins are represented here, and quite a few proteins involved in extracellular matrix agglutination, such as lectin, collagen, EF-Hand, thrombospondin and fibrinogen. ...
Background:
Despite recent work to characterize gene expression changes associated with larval development in oysters, the mechanism by which the larval shell is first formed is still largely unknown. In Crassostrea gigas, this shell forms within the first 24 h post fertilization, and it has been demonstrated that changes in water chemistry can cause delays in shell formation, shell deformations and higher mortality rates. In this study, we use the delay in shell formation associated with exposure to CO2-acidified seawater to identify genes correlated with initial shell deposition.
Results:
By fitting linear models to gene expression data in ambient and low aragonite saturation treatments, we are able to isolate 37 annotated genes correlated with initial larval shell formation, which can be categorized into 1) ion transporters, 2) shell matrix proteins and 3) protease inhibitors. Clustering of the gene expression data into co-expression networks further supports the result of the linear models, and also implies an important role of dynein motor proteins as transporters of cellular components during the initial shell formation process.
Conclusions:
Using an RNA-Seq approach with high temporal resolution allows us to identify a conceptual model for how oyster larval calcification is initiated. This work provides a foundation for further studies on how genetic variation in these identified genes could affect fitness of oyster populations subjected to future environmental changes, such as ocean acidification.
... In later larval and juvenile stages, calcification is performed by the mantle tissue. Following settlement and metamorphosis, the mineralogy of the shell changes: while veliger prodissoconch I and II are exclusively composed of amorphous and aragonitic CaCO 3 (Medakovic, 2000;Weiss et al., 2002;Weiss and Schönitzer, 2006), the newly formed shell of juveniles consists of calcite, which is a more stable polymorph (Medakovic et al., 1997). Nevertheless, this shift to a more stable polymorph does not seem to cause higher tolerance of the calcification process itself to adverse carbonate chemistry. ...
Bivalve calcification, particularly of the early larval stages, is highly
sensitive to the change in ocean carbonate chemistry resulting from
atmospheric CO2 uptake. Earlier studies suggested that declining
seawater [CO32−] and thereby lowered carbonate saturation affect
shell production. However, disturbances of physiological processes such as
acid-base regulation by adverse seawater pCO2 and pH can affect
calcification in a secondary fashion. In order to determine the exact
carbonate system component by which growth and calcification are affected it
is necessary to utilize more complex carbonate chemistry manipulations. As
single factors, pCO2 had no effects and [HCO3-] and pH had only limited
effects on shell growth, while lowered [CO32−] strongly impacted
calcification. Dissolved inorganic carbon (CT) limiting conditions led
to strong reductions in calcification, despite high [CO32−],
indicating that [HCO3-] rather than [CO32−] is the
inorganic carbon source utilized for calcification by mytilid mussels.
However, as the ratio [HCO3-] / [H+] is linearly correlated
with [CO32−] it is not possible to differentiate between these
under natural seawater conditions. An equivalent of about 80 μmol kg−1 [CO32−] is required to saturate inorganic carbon supply
for calcification in bivalves. Below this threshold biomineralization rates
rapidly decline. A comparison of literature data available for larvae and
juvenile mussels and oysters originating from habitats differing
substantially with respect to prevailing carbonate chemistry conditions
revealed similar response curves. This suggests that the mechanisms which
determine sensitivity of calcification in this group are highly conserved.
The higher sensitivity of larval calcification seems to primarily result
from the much higher relative calcification rates in early life stages. In
order to reveal and understand the mechanisms that limit or facilitate
adaptation to future ocean acidification, it is necessary to better
understand the physiological processes and their underlying genetics that
govern inorganic carbon assimilation for calcification.
... Mytilus EP (Extrapallial Protein), an acidic calcium binding protein, regulates the production of different polymorphs of calcium carbonate (Yin et al., 2009). Both EP and CA showed the highest expression in D-veligers, supporting the results from SEM analyses, showing that the formation of a complete normal shell occurred only at 48 h pf, and confirming the key physiological role of both transcripts in initial biomineralization (Medakovic, 2000;Yin et al., 2009Yin et al., , 2005. Exposure of fertilized eggs to 1 mg/L BPA resulted in small but significant up-regulation of most transcripts. ...
... This CA may help catalyzing the supply of inorganic carbon in the calcifying medium. As in other metazoans' calcium carbonate biominerals, such as calcareous sponges spicules [19], demosponges basal skeletons [21], molluscan shells [24,25] and statocyts [26], sea urchin tests and spines [25,27], CAs have also been found in the organic matrices of coral skeletons [28][29][30][31][32][33]. These CAs could play a key role in the precipitation of calcium carbonate. ...
Although the ability to elaborate calcium carbonate biominerals was apparently gained independently during animal evolution, members of the alpha carbonic anhydrases (α-CAs) family, which catalyze the interconversion of CO2 into HCO3-, are involved in the biomineralization process across metazoans. In the Mediterranean red coral Corallium rubrum, inhibition studies suggest an essential role of CAs in the synthesis of two biominerals produced in this octocoral, the axial skeleton and the sclerites. Hitherto no molecular characterization of these enzymes was available. In the present study we determined the complete set of α-CAs in C. rubrum by data mining the genome and transcriptome, and measured their differential gene expression between calcifying and non-calcifying tissues. We identified six isozymes (CruCA1-6), one cytosolic and five secreted/membrane-bound among which one lacked two of the three zinc-binding histidines and was so referred to as a carbonic anhydrase related protein (CARP). One secreted isozyme (CruCA4) showed specific expression both by qPCR and western-blot in the calcifying tissues, suggesting its involvement in biomineralization. Moreover, phylogenetic analyses of α-CAs, identified in six representative cnidarians with complete genome, support an independent recruitment of α-CAs for biomineralization within anthozoans. Finally, characterization of cnidarian CARPs highlighted two families: the monophyletic cytosolic CARPs, and the polyphyletic secreted CARPs harboring a cnidarian specific cysteine disulfide bridge. Alignment of the cytosolic CARPs revealed an evolutionary conserved R-H-Q motif in place of the characteristic zinc-binding H-H-H necessary for the catalytic function of α-CAs.
... Transport of ions by the gills and the mantle is an essential function for shell formation. Although the process of shell development is a wellstudied subject in molluscs (Kniprath, 1980;Eyster, 1983;Medaković, 2000;Hohagen and Jackson, 2013), understanding of the calcium transport mechanisms is not comprehensive. The general view of bivalve shell formation suggests that biomineralisation occurs via precipitation of CaCO 3 crystals in the organic matrix from a supersaturated solution of CaCO 3 in the extrapallial space i.e. the compartment between the outer mantle epithelium (OME) and the shell (Wheeler and Sikes, 1984;Levi-Kalisman et al., 2001;Joubert et al., 2010;Marie et al., 2011;Marin et al., 2012). ...
Shell growth of oysters requires calcium uptake from the environment and transport to the area of shell formation. A shell regeneration assay in combination with radiolabelled calcium was used to investigate uptake and distribution of calcium to different tissues and hemolymph fractions in Pacific oysters, Crassostrea gigas (Bivalvia, Ostreoida). Oysters were notched at the shell margin and subsequently sampled for hemolymph and grading of shell regeneration during a two week experimental period. Half of the oysters were additionally exposed to 45Ca and sampled for hemolymph and tissues. Total plasma calcium concentrations increased in notched oysters compared to controls on 1, 2 and 7 days after notching. A decrease in plasma calcium levels was apparent on day 4, for both total and ionic calcium. The shell regeneration assay in the notched oysters resulted in a visible deposition of CaCO3 onto the regenerate from day 7 onwards. This was coinciding with an increased uptake of total calcium on days 11 and 14 as well as free, i.e. ionic and ligand-bound calcium, on day 14. At day 1, notching also increased calcium uptake into the mantle tissues, in areas above the notch and near the hinge. During the experiment, both the total hemocyte count and the number of granulocytes increased in notched compared to control oysters. The present study suggests that induced shell damage results in a dynamic regulation of the calcium uptake from the environment and the distribution of calcium within the body, starting directly after notching. Increases in both total calcium concentrations and uptake rates coincided with the visible depositions of CaCO3 on the regenerate shell. C. gigas was found to transport calcium mainly in the ionic form in the hemolymph, with only minor parts being bound to proteins or smaller ligands. Hemolymph measurement also revealed that C. gigas is able to regulate the extracellular concentrations of calcium and potassium. The changes in plasma calcium concentrations and speciation, concomitant with increases in granulocytes indicate that multiple calcium transport processes are activated after induced shell damage.
... Interestingly, other unionoid nacre proteins, derived from E. complanata incomplete sequence of contig_3333 and _23650, present also CBD and LamG domains and their amino acid sequences This enzyme is believed to be essential for biomineral formation since bicarbonate, the product of the catalytic process, can directly react with calcium ions to form calcium carbonate [28]. Furthermore, CA has been found in the organic matrices of various metazoan skeletons [29][30][31]. ...
The formation of the molluscan shell nacre is regulated to a large extent by a matrix of extracellular macromolecules that are secreted by the shell forming tissue, the mantle. This so called “calcifying
matrix” is a complex mixture of proteins and glycoproteins that is assembled and occluded within
the mineral phase during the calcification process. While the importance of the calcifying matrix to
shell formation has long been appreciated, the molecular basis that dictates nacre formation remains
largely uncharacterized.
Recent expressed sequence tag (EST) investigations of the freshwater mussels (Elliptio complanata
and Villosa leinosa) provide an opportunity to further characterize the proteins in the bivalve shell
by a proteomic approach. In this study, we have identified a total of 15 proteins from their nacre
insoluble matrices. Few of these proteins, such as Pif, MSI60, Nacrein-like, Shematrin, Kunitz-like
containing, Papilin-like, LamG containing, Chitin-binding containing, M-rich and Q-rich proteins,
appear to be analogs, if not true homologs, of proteins previously described from the pearl oyster or
the edible mussel nacre matrices. This work constitutes a comprehensive EST-based nacre
proteomic study of non-pteriomorphid bivalves that concretely gives us the opportunity to describe
the molecular basis of deeply conserved nacre biomineralization toolkit within nacreous shell
bearing bivalves.
... Calcium crystals are laid down during the shell repair 348 process (Trinkler et al, 2011). Moreover, CA activity was not associated to shell growth in mussels (Medakovic et al, 2000). These results suggested an involvement 350 of CA in the formation of conchiolin deposit which is often associated to the nacrezation process (Cheng, 1967). ...
The culture of Manila clams in Brittany has faded from the 80 years after the onset of the disease called vibriosis of the Brown Ring (MAB). This disease is caused by the presence of the bacterium Vibrio tapetis causes a characteristic conchiolin deposit on the inner edge of the valves of the clams. The animals are able to repair the shell at the conchiolin deposits by a phenomenon of recalcification. Broodstock was selected according to their stage of disease development after inoculation of bacteria into the pallial cavity or following their physiological response in the hemolymph. Families of clams were produced and comparisons of the response to the disease were made between families in order to differentiate with the least susceptible individuals to this disease. Thus, these studies have to distinguish two families showing different characteristics of the immune level and hemocyte parameters in the development of the MAB. A relationship between families and ornamentation/color of the shell has been detected and enabled, in the thesis, to differentiate families in addition to the microsatellite tool. Moreover, it was shown that the V. tapetis induces production of nitric oxide in vitro in hemocytes of Manila clams. Moreover, when NO production is inhibited, the number of adherent cells decreases. Finally, molecular approaches have helped to highlight the specific expression of two gene transcripts (carbonic anhydrase like and serpin) in the mantle related to the development of the MAB.
... In later larval and ju- venile stages, calcification is performed by the mantle tissue. Following settlement and metamorphosis, the mineralogy of the shell changes: while veliger prodissoconch I and II are exclusively composed of amorphous and aragonitic CaCO 3 (Medakovic, 2000;Weiss et al., 2002;Weiss and Schönitzer, 2006), the newly formed shell of juveniles consists of calcite, which is a more stable polymorph ( Medakovic et al., 1997). Nevertheless, this shift to a more stable polymorph does not seem to cause higher tolerance of the calcification process it- self to adverse carbonate chemistry. ...
Impact of seawater carbonate chemistry on the calcification
of marine bivalves
... The presence of CA in vertebrate hard tissues which bare minerals of calcium phosphate, is linked to the supply of carbonate ions which become incorporated into the initial mineralization site and initiate the nucleation (Kakei et al., 1996). Similarly, CA is believed to be involved in the biomineralization process via the bicarbonate or carbonate supply in the sea urchin (Mann et al., 2008; Livington et al., 2006; Hofmann et al., 2008), in Mollusca (Medakovic, 2000; Yu et al., 2006); Marie et al., 2008), in Cnidaria (Kingsley and Watabe, 1987, Ip et al., 1991) and in the gland responsible for eggshell formation in birds (Gay and Mueller, 1973). In contrast, in marine sponges, the enzyme silicase belonging to the family of CAs (Müller et al., 2007; Kirill et al., 2011), provides silica depolymerization in a mechanism similar to zinc-dependent metalloenzymes hydrolyzing ethers (Schroeder et al., 2007, Wang et al., 2012) In bones, calcium phosphate is resorbed by osteoclasts. ...
This study is focused on the Mediterranean sea urchin species, Paracentrotus lividus (P. lividus). The major part of the study covered the characterization of genes and proteins involved in biomineralization of P. lividus embryo and adult. The aim was to establish P. lividus as a working model for biomineralization studies. The primary goal was to develop a set of molecular tools involving plasmids, labeling probes, recombinant proteins and antibodies which subsequently were used in developmental and biomineralization studies. Candidate genes were selected based on previous findings in other species and on in silico analysis and comparison to the invertebrates and the sea urchin. The selected biomolecules involved acidic proteins (p16, p19), lectins (advillin, sm30α, sm50, galectin-8), a signaling protein (tetraspanin) and an enzyme (carbonic anhydrase). All the above groups of encoded proteins are known to participate in biomineralization in various species and therefore they were selected for the study of their role in the formation of the sea urchin skeleton.
The protein-coding mRNA sequences p16, p19, advillin, tetraspanin, sm30α, sm50, carbonic anhydrase and galectin-8 were identified and cloned, following molecular biology techniques. The putative protein domains were analyzed and a phylogenetic comparison among the sea urchin species of P. lividus, Strongylocentrotus purpuratus (S. purpuratus) and Lytechinus variegatus (L. variegatus) revealed their evolution relation. Furthermore, the localization of the temporal and spatial expression of each transcript throughout the embryo development was characterized by comparative qPCR and whole mount in situ hybridization (WMISH). The acquired experimental data, revealed the expression profile of each one of these genes in the developing embryo and the insights on the role of each gene in development and skeletogenesis were discussed. Additionally, a functional characterization of recombinant carbonic anhydrase and galectin-8 proteins, cloned and expressed in vitro in E. coli and purified by affinity chromatography, gave insights of the role of each protein in development. Specific polyclonal antibodies were prepared and used to identify by Western Blot and ELISA, Pl-CA and Pl-Galectin-8-like proteins, in both the embryo and in the occluded matrix proteins of adult P. lividus, showing the importance of the two proteins in development. Functional assays, involving the in vitro esterase activity of Pl-CA and the lactose-specific hemagglutination activity of the lectin Pl-GALECTIN-8, confirmed that both recombinant proteins were active. Additionally, the biological role of Pl-GALECTIN-8 in cell adhesion was tested in vivo, in a human cell adhesion assay.
Furthermore, aiming towards a complete characterization of the biomineralization proteins both in the embryo and in adult, the proteome of the occluded matrix proteins from adult P. lividus mineralized tests, was examined. Proteins were extracted from purified calcareous tests and identified by Liquid Chromatography coupled with Mass Spectrometry (LC-MS/MS). For the first time, the effect of the sea urchin occluded matrix protein on the formation of calcite is studied by an in vitro crystallization assay.
... Cruise Sal TA (μmol kg −1 ) DIC (μmol kg −1 ) TOxN (μmol l −1 ) Si (μmol l −1 ) PO 4 (μmol l −1 ) pH pCO 2 (μatm) (Medaković 2000). Since aragonite is 50 % more soluble than calcite, these aragonitic larval stages of molluscs are expected to be more sensitive to ocean acidification than calcitic organisms. ...
The wintertime spatial distribution of carbonate parameters in outer estuarine and coastal waters around Ireland is described from total alkalinity (TA) and dissolved inorganic carbon (DIC) data collected between 2010 and 2013. Due to predominantly limestone bedrock of their river catchments, the River Shannon and Barrow, Nore and Suir River system export high concentrations (>3800 μmol kg−1) of TA to their estuarine and inshore coastal waters where estuarine alkalinity decreases with increasing salinity. TA is lower in rivers with a non-calcareous bedrock, with positively correlated alkalinity-salinity relationships in both the Lee and Foyle outer estuaries. Winter pCO2 in the Shannon, Barrow/Nore/Suir and Lee estuaries is supersaturated relative to atmospheric CO2, while pCO2 in the outer Liffey estuary is slightly lower than atmospheric CO2 in three consecutive winters, indicative of a CO2 sink. Winter pCO2 is close to atmospheric equilibrium along the western shelf and through the centre of the Irish Sea, while it is a CO2 sink across the North Channel. While aragonite was supersaturated in most Irish waters, it was close to undersaturation in both the Lee estuary, attributed to its low alkalinity freshwater source, and Barrow/Nore/Suir estuary related to the flux of high concentrations of DIC from this river system. The seasonal impacts on inorganic carbon chemistry was also investigated by comparing winter and summer data collected between 2009 and 2013 along two transects in western coastal waters and along the western shelf edge. DIC was ~60 μmol kg−1 lower in summer relative to winter in the coastal transects and 39 μmol kg−1 lower along the shelf edge, accompanied by depleted nutrients and supersaturation of dissolved oxygen during summer, indicative of primary production. TA was generally higher in summer relative to winter corresponding with a decrease in nitrate, indicating that primary production dominated the TA distribution over calcification. An exception to this was at two stations along the shelf edge where TA was lower in summer relative to winter (51 μmol kg−1) and coincides with high reflectance in satellite images from a coccolithophore bloom at the time of sampling. While pCO2 was close to atmospheric equilibrium along the shelf edge during winter, this area was a CO2 sink during summer, apart from the stations where calcification was likely occurring resulting in elevated CO2 relative to atmospheric concentrations.
... Additionally, 0Á1 to 0Á7 wt% quartz is present in shells cultured in the aquaria settings. The origin of this quartz phase is debated (Medakovic, 2000) and it might either be incorporated into molluscan shell layers during rapid shell growth or it is a result of some disturbance in the biomineralization processes. ...
Throughout much of Earth's history, marine carbonates have represented one of the most important geological archives of environmental change. Several pivotal events during the Phanerozoic, such as mass extinctions or hyperthermal events have recently been associated with ocean acidification. Nevertheless, well-defined geological proxies for past ocean acidification events are, at best, scarce. Here, experimental work explores the response of bivalve shell ultrastructure and isotope geochemistry (δ13C, δ18O and δ26Mg) to stressful environments, in particular to sea water acidification. In this study, the common blue mussel, Mytilus edulis, was cultured (from early juvenile stages to one year of age) at four pH regimes (pHNBS 7·2 to pH 8·0). Shell growth rate and ultrastructure of mainly the calcitic portion of the shells were compared between experimental treatments. Specimens exposed to low-pH environments show patches of disordered calcitic fibre orientation in otherwise well-structured shells. Furthermore, the electron backscattered diffraction analyses reveal that, under acidified conditions, the c-axis of the calcite prisms exhibits a bimodal or multi-modal distribution pattern. Similar shell disorder patterns have been reported from mytilids kept under naturally acidified sea water conditions. In contrast, this study found no evidence that different pH regimes affect shell carbon, oxygen or magnesium isotope ratios. Based on these observations, it is proposed that: (i) stressful environments, in this case low sea water pH, predictably affect bivalve biomineralization patterns; and (ii) these findings bear potential as a novel (petrographic) proxy for ancient sea water acidification. An assessment of the applicability of these data to well-preserved fossil shell material from selected time intervals requires additional work.
... Additionally, 0Á1 to 0Á7 wt% quartz is present in shells cultured in the aquaria settings. The origin of this quartz phase is debated (Medakovic, 2000) and it might either be incorporated into molluscan shell layers during rapid shell growth or it is a result of some disturbance in the biomineralization processes. ...
Throughout much of Earth’s history, marine carbonates have represented
one of the most important geological archives of environmental change. Several
pivotal events during the Phanerozoic, such as mass extinctions or
hyperthermal events have recently been associated with ocean acidification.
Nevertheless, well-defined geological proxies for past ocean acidification
events are, at best, scarce. Here, experimental work explores the response of
bivalve shell ultrastructure and isotope geochemistry (d13C, d18O and d26Mg)
to stressful environments, in particular to sea water acidification. In this
study, the common blue mussel, Mytilus edulis, was cultured (from early
juvenile stages to one year of age) at four pH regimes (pHNBS 7�2 to pH 8�0).
Shell growth rate and ultrastructure of mainly the calcitic portion of the
shells were compared between experimental treatments. Specimens exposed
to low-pH environments show patches of disordered calcitic fibre orientation
in otherwise well-structured shells. Furthermore, the electron backscattered
diffraction analyses reveal that, under acidified conditions, the c-axis of the
calcite prisms exhibits a bimodal or multi-modal distribution pattern. Similar
shell disorder patterns have been reported from mytilids kept under naturally
acidified sea water conditions. In contrast, this study found no
evidence that different pH regimes affect shell carbon, oxygen or magnesium
isotope ratios. Based on these observations, it is proposed that: (i) stressful
environments, in this case low sea water pH, predictably affect bivalve biomineralization
patterns; and (ii) these findings bear potential as a novel (petrographic)
proxy for ancient sea water acidification. An assessment of the
applicability of these data to well-preserved fossil shell material from
selected time intervals requires additional work.
Pinctada fucata martensii and P. maxima are two main traditional pearl oyster species that can produce seawater pearls. Our previous study showed a higher clearance rate (CR) and growth performance in P. f. martensii than in P. maxima fed with Isochrysis galbana. In this study, the P. f. martensii and P. maxima juveniles of two sizes (large and small) were fed with six different microalgae diets [I. galbana (I), Platymonas subcordiformis (P), Chaetoceros muelleri I, I+P, I+C, and P+C] to evaluate the differences in growth, feeding, and metabolism between two pearl oyster species. After 60 d of the rearing period, P. f. martensii and P. maxima fed with mixed microalgae showed a significantly higher relative growth rate (RGR) than those fed with single microalgae (P< 0.05). The RGRs were significantly higher in P. f. martensii than those in P. maxima fed with the same diets (P< 0.05). The RGRs showed a decreasing tendency with the growth in both pearl oyster species. The CRs of pearl oysters fed with mixed microalgae were significantly higher than those fed with single microalgae (P< 0.05), and the CRs of P. f. martensii were significantly higher than those of P. maxima fed with the same diets (P< 0.05). Significantly lower respiration rates (RRs) were observed in small-size P. f. martensii groups fed with I, P, and I+P diets and all large P. f. martensii groups compared to P. maxima fed with the same diets (P< 0.05). Higher activities of amylase, cellulase, lipase, and pepsin in P. f. martensii were observed compared to P. maxima fed with the same diets at two sizes. The pepsin activities in P. maxima decreased with the growth, while there were no consistent pepsin activities of P. f. martensii with the growth. The carbonic anhydrase activities in P. maxima were significantly higher than those in P. f. martensii fed with the same diets (P< 0.05). The carbonic anhydrase activities were highest in the I+C diet group, followed by C+P and I+P, I, C, and P groups. Significant differences were observed among different diet groups in the same pearl oyster species (P< 0.05). Our results suggest that the lower CR and activities of digestive enzymes and higher RRs and activities of carbonic anhydrase may cause a lower growth rate of P. maxima compared to P. f. martensii.
The increasing presence of anthropogenic contaminants in the environment may constitute a challenge to non-target biota, considering that most contaminants can exert deleterious effects. Salicylic acid (SA) is a non-steroid anti-inflammatory drug (NSAID) which exerts its activity by inhibiting the enzyme cyclooxygenase (COX). Another class of drugs is that of the diuretics, in which acetazolamide (ACZ) is included. This pharmaceutical acts by inhibiting carbonic anhydrase (CA), a key enzyme in acid-base homeostasis, regulation of pH, being also responsible for the bio-availability of Ca²⁺ for shell biomineralization processes. In this work, we evaluated the chronic (28-day) ecotoxicological effects resulting from the exposures to SA and ACZ (alone, and in combination) on individuals of the marine mussel species Mytillus spp., using enzymatic (catalase (CAT), glutathione S-transferases (GSTs), COX and CA), non-enzymatic (lipid peroxidation, TBARS levels) and morphological and physiological (shell hardness, shell index and feeding behaviour) biomarkers. Exposure to ACZ and SA did not cause significant alterations in CAT and GSTs activities, and in TBARS levels. In terms of CA, this enzyme was inhibited by the highest concentration of ACZ in gills of exposed animals, but no effects occurred in the mantle tissue. The activity of COX was not altered after exposure to the single chemicals. However, animals exposed to the mixture of ACZ and SA evidenced a significant inhibition of COX activity. Morphological and physiological processes (namely, feeding, shell index, and shell hardness) were not affected by the here tested pharmaceutical drugs. Considering the general absence of adverse effects, further studies are needed to fully evaluate the effects of these pharmaceutical drugs on alternative biochemical and physiological pathways.
Three cohorts of Pacific oyster (Crassostrea gigas) larvae at Whiskey Creek Shellfish Hatchery (WCH) in Netarts Bay, Oregon, were monitored for stable isotope incorporation and biochemical composition: one in May 2011 and two in August 2011. Along with measures of growth and calcification, we present measurements of stable isotopes of carbon in water, algal food, and the shell and tissue, and nitrogen in food and tissue across larval development and growth. These relatively unique measures through larval ontogeny allow us to document isotopic shifts associated with initiation and rate of feeding, and the catabolism of C-rich (lipid) and N-rich (protein) pools. Similar ontological patterns in growth and bulk composition among the cohorts reinforce prior results, suggesting that the creation of the initial shell is energetically expensive, that the major carbon source is ambient dissolved inorganic carbon, and that the major energetic source during this period is maternally derived egg lipids. The May cohort did not isotopically reflect its food source as rapidly as the August cohorts, indicating slower feeding and/or higher catabolism versus anabolism. Our measurements also document differences in bulk turnover of organic carbon and nitrogen pools within the larvae, showing far greater conservation of nitrogen than carbon. These stable isotope and bulk biochemical measurements appear to be more sensitive indicators of sub-lethal environmental stress than the commonly used metrics of development and growth.
Carbonic anhydrases (CA: carbonate hydrolase: E.C.4.2.1.1) from leaves and roots of mature potato (Solatium tuberosum) were purified and characterized. The purification levels of enzymes were 87.91 fold and 40.601 fold in leaves and roots, respectively. The optimum temperature was 40 °C in leaves and in roots. pHs optimal were 8.S and 11 in leaves and in roots, respectively. Enzymes of leaves were formed 5 monomers that it's having molecular weights of 22,000, 28,000, 35,000, 40,000, 65,000, 5 polymers that it's having 73,000, 80,000, 83,000,132,000 and 200,000 Dalton and these proteins had carbonic anhydrase activity. But there is at the levels of 22,000,28,000,35,000 and 132,000 Dalton in gel filtration, for leaves. SDS-PAGE was done for roots and leaves and subunits were obtained. In addition the enzyme against the effect of NaN 3, KSCN and sulphanylamide, which was known as an inhibitor of mammalian carbonic anhydrase, was determined. Potato (Solanum tuberosum) has ascorbic acid (vitamin C) in very high level and iron and calcium ions in low level. So, the effect of ascorbic acid, FeCl3 and CaCl2 in different concentrations on enzyme was determined for roots and leaves, separately. Finally, carbonic anhydrase was purified from roots and leaves of potato (Solanum tuberosum), separately and they were done optimal. Carbonic anhydrase functions in respiration and has a part in photosynthesis in plants. It was a serious deficiency that carbonic anhydrase wasn't defined in potato (Solanum tuberosum).
Carbonic anhydrase (CA) is involved in ion transport, acid-base balance and pH regulation by catalyzing the interconversion of CO2 and HCO3(-). In this study, full-length cDNA sequences of two CA isoforms were identified from Portunus trituberculatus. One was Portunus trituberculatus cytoplasmic carbonic anydrase (PtCAc) and the other one was Portunus trituberculatus glycosyl-phosphatidylinositol-linked carbonic anhydrase (PtCAg). The sequence of PtCAc was formed by an ORF of 816bp, encoding a protein of 30.18kDa. The PtCAg was constituted by an ORF of 927bp, encoding a protein of 34.09kDa. The deduced amino acid sequences of the two CA isoforms were compared to other crustacean(') CA sequences. Both of them reflected high conservation of the residues and domains essential to the function of the two enzymes. The tissue expression analysis of PtCAc and PtCAg were detected in gill, muscle, hepatopancreas, hemocytes and gonad. PtCAc and PtCAg gene expressions were studied under salinity and pH challenge. The results showed that when salinity decreased (30 to 20ppt), the mRNA expression of PtCAc increased significantly at 24 and 48h, and the highest value appeared at 24h. The mRNA expression of PtCAg had the same situation with PtCAc. However, when salinity increased (30 to 35ppt), only the mRNA expression of PtCAc increased significantly at 48h. When pH changed, only the mRNA expression of PtCAc increased significantly at 12h, which was under low pH situation. The mRNA expression of PtCAg increased significantly at 12-48h, and there was no significant difference of the expression between the pH challenged group and the control group in other experimental time. The results provided the base of understanding CA' function and the underlying mechanism in response to environmental changes in crustaceans.
Bivalve calcification, particularly of the early larval stages, is highly sensitive to the change in ocean carbonate chemistry resulting from atmospheric CO2 uptake. Earlier studies suggested that declining seawater [CO32−] and thereby lowered carbonate saturation affect shell production. However, disturbances of physiological processes such as acid-base regulation by adverse seawater pCO2 and pH can affect calcification in a secondary fashion. In order to determine the exact carbonate system component by which growth and calcification are affected it is necessary to utilize more complex carbonate chemistry manipulations. As single factors, pCO2 had no effects and [HCO3-] and pH had only limited effects on shell growth, while lowered [CO32−] strongly impacted calcification. Dissolved inorganic carbon (CT) limiting conditions led to strong reductions in calcification, despite high [CO32−], indicating that [HCO3-] rather than [CO32−] is the inorganic carbon source utilized for calcification by mytilid mussels. However, as the ratio [HCO3-] / [H+] is linearly correlated with [CO32−] it is not possible to differentiate between these under natural seawater conditions. An equivalent of about 80 μmol kg−1 [CO32−] is required to saturate inorganic carbon supply for calcification in bivalves. Below this threshold biomineralization rates rapidly decline. A comparison of literature data available for larvae and juvenile mussels and oysters originating from habitats differing substantially with respect to prevailing carbonate chemistry conditions revealed similar response curves. This suggests that the mechanisms which determine sensitivity of calcification in this group are highly conserved. The higher sensitivity of larval calcification seems to primarily result from the much higher relative calcification rates in early life stages. In order to reveal and understand the mechanisms that limit or facilitate adaptation to future ocean acidification, it is necessary to better understand the physiological processes and their underlying genetics that govern inorganic carbon assimilation for calcification.
The acidification of oceans is predicted to fundamentally alter marine ecosystems. Previous studies have found that elevated CO 2 has an effect on adult calcification, fertilisation and larval development, perhaps because of the organisms' inability to regulate acid-base status, but little is known about the mechanisms that underlie such responses. This study investigated the growth response of larvae of a wild and selectively bred line of the Sydney rock oyster, Saccostrea glomerata, to elevated CO 2 and measured the pattern of expression of proteins. Overall exposure to elevated CO 2 caused a significant reduction in the shell length of D-veliger larvae of the wild, but not in the selectively bred line. Prior to this study, differences in growth between selectively bred and wild oysters have only been found following settlement Proteome analysis of D-veliger larvae using two-dimensional gel electrophoresis detected a significantly greater number of protein spots in selectively bred compared to wild oyster lines. In addition, a comparison of the proteins expressed between selectively bred and wild larvae exposed to elevated CO 2 and ambient conditions showed that a number of proteins were up- or down-regulated and in some cases, switched on at elevated CO 2 in selectively bred lines, but not found in the wild lines. Identification of these differentially expressed proteins may assist to "climate proof" of important aquacultural industries.
The accumulation of carbon dioxide (CO2) is the main cause of global climate change and it is very important to develop technologies that can reduce atmospheric CO2 level. Although several physical or chemical CO2 capture processes are being developed, the efficiency of capture/sequestrationshould be increased. Carbonic anhydrase (CA, EC 4.2.1.1), a metalloenzyme, has been considered as an important biocatalyst for CO2 capture system development because CA has the highest efficiency of CO2 conversion via hydration (CO2 + H2O ↔ HCO3− + H+, kcat: ∼106 s−1). In this review, biocatalytic mechanism and functional properties of CA are reviewed in terms of its distinctive CO2 converting ability. In addition, recent applications of several CAs are presented in CO2 capture process development. This review would present guidelines for scientists and researchers to understand CA functions and develop interest in the practical applications of CA, in particular, as biocatalytic agent for more efficient CO2 capture process development.
Biological organisms produce organic-inorganic nanocomposite composites that are hierarchically organized in composition and microstructure, containing both inorganic and organic components in complicated mixtures. The process related to the generation and regeneration of organic-inorganic complex in nature is called biomineralization process. Understanding how the process operates in a biological environment is a valuable guide to the synthesis of novel advanced material and developing important industrial processes. Like the mechanism of organisms, mollusks were also synthesized from interaction between organic matrices and minerals and their morphology was designed through biomineralization. In this study, shell formation has been studied as a bio-model and the application of biomimetics based on biomineralization is focused.
Metal content in samples of shell of mussel Mytilus galoprovincialis was determined by inductively coupled plasma atomic emission spectrometry (ICP-AES). The efficiency of conversion of crude samples into solution by acid digestion in an open plate and in a microwave oven was examined by use of certified reference material of marine sediment and laboratory made standards of calcite and aragonite. Influence of high Ca content matrix on emission intensities of Al, Ba, Cd, Cu, Fe, Mg, Mn, Na, Ni, Pb, Sr and Zn was observed as depression of emission signal for most of the measuring elements, ranging from 0.8% to 8%. Greater values were noted at Ba and Ni emission lines. Enhancement of signals was observed for Na and Mg lines. The determination of As, Sb, Se and Sn was performed by HG/ICP-AES. The greater abundance of Sn was found in samples collected near the Al-processing industry centre. No detectable concentrations of As, Sb, and Se were found in shell samples. Results of ICP-AES metal analysis showed that samples collected near harbours, city waste or sewage outlets, and chemical industry centres indicate the certain level of contamination. It is shown that shell analysis provides useful data in determination of marine environment status.
The published evidence of impacts of ocean acidification and on marine
calcifiers has emphasized the need to understand the molecular
mechanisms of biomineralisation. Crassostrea gigas is an ideal organism
to examine these processes as: 1) the hatchery rearing of larval stages
is well constrained, 2) studies have established an ontogenetic switch
in deposition of carbonate polymorphs from aragonite in larval shells to
calcite in adults and 3) it is a globally-important commercial species.
Research summarized in this presentation will identify some of the
molecular mechanisms involved in calcification processes during ontogeny
of Crassostrea gigas, as well as possible impacts of changes in
environmental conditions such as temperature and pH. Data will be
presented from a quantitative real-time PCR study of the changes in gene
expression during development in different environments. Additionally
scanning electron microscopy and infrared spectroscopy analyses of shell
microstructures and composition will be summarised to correlate changes
in gene expression with end-point differences in shell structure.
Preliminary results suggest that changes in the environmental conditions
lead to differences in expression patterns of genes involved in
biomineralisation processes. The combined effects of ambient seawater
temperature and low pH show the greatest negative effect on larval shell
development, identified as malformations, eroded shell surfaces and a
significant decrease in shell size. However, the effect of higher
seawater temperature seems to amend the effects of ocean acidification
on larval shell development.
The combined effect of both carbonic anhydrase (CA) and the rigidity of polyethylene glycol (PEG) were found to assist the bio-mineralized crystallization behavior of CO2 differentially. In this study, different forms of magnetically responsive calcium carbonate (CaCO3) crystal composites were successfully formed from gaseous CO2 by using the different forms of polyethylene glycols (PEGs) in a constant CO2 pressure controlled chamber. Polygonal particles were produced with more rigid polymer chains (branched PEG), whereas less rigid polymer chains (PEG) induced the formation of ellipsoidal particles. However, no morphological changes occurred without the presence of CA.
The doping method in quantitative X-ray diffraction phase analysis is described. The method involves the addition to the investigated system of known amounts of the components, the weight fractions of which are to be simultaneously determined. The weight fraction of a component is related to the intensities diffracted by that component and by any non-added component, before and after doping. The method can also be applied to systems containing unidentified components by analysing for only those components of interest, as well as for amorphous-content determination.
A specific case of the doping method [Popovic & Gržeta-Plenkovic (1979). J. Appl. Cryst.12, 205–208] is elaborated, in which the weight fraction of a crystalline component in the multicomponent system can be determined from the measurement of diffraction line intensities of that component only. Optimum conditions to attain maximum accuracy of the doping method are discussed.
Publisher Summary This chapter focuses on the rearing of bivalve mollusks. The rearing of larval and juvenile bivalves requires an adequate supply of sea water of proper salinity and free of substances that may interfere with their normal development. To condition mollusks for out-of-season spawning, it is necessary to keep them in running sea water at temperatures of 18°C to 20°C or sometimes higher. Warm sea water is also needed for rearing larvae and juveniles during the cold season. Conditioning of bivalves to develop mature gonads during the cold part of the year is relatively simple. It consists of placing mollusks, brought from their natural environment where water temperature may be near freezing, into somewhat warmer water, and then gradually increasing the temperature several degrees each day until the desired level is reached. Development of the egg of a bivalve is also discussed and the rearing of different species like Crassostrea virginica , Modiolus demissus , and Crassostrea gigas is reviewed.
H2CO3 (Berliner and Orloff, 1956) and has been shown to accelerate both deposition and dissolution of calcium carbonate in vitro (Meldrum and Roughton, 1933). In view of its possible implication in shell formation, the mantle tissues of molluscs have been examined and the enzyme demonstrated in several of them (Florkin and de Marchin, 1941; Maetz, 1946; Freeman and Wilbur, 1948; Wilbur and Anderson, 1950; Stolkowski, 1951; Tsujii and Machida, 1953; Ikinago, 1954; Kawai, 1954a, 1954b; Larraneta and Ponz, 1954; Clark, 1957). However, among 20 species examined, Freeman and Wilbur (1948) found little or no carbonic anhydrase activity in mantle tissue in two, a pelecypod and a gastropod, both shell-formers, and Stolkowski (1951) found none in the mantles of Ostrea edulis and several other shell-forming molluscs. Such results suggest that at least in some species the enzyme is not essential for shell formation. Stolkowski (1951) apparently first used a carbonic anhydrase inhibitor, benzenesulfonamide, in studies of the role of the enzyme in molluscs, observing decreased shell regeneration in Helix aspersa in the presence of the drug. He concluded that the enzyme is important in determining the crystalline form in which calcium carbonate appears in the shell. Wilbur and Jodrey (1955) found that two carbonic anhydrase inhibitors, 2-benzothiazolesulfonamide and Diamox (2-acetylamino-1,3,4-thiadiazole-5-sulfonamide), markedly decrease the rate of de posit of Ca45 in the shell of Crassostrea virginica. Abolins-Krogis (1958) in a study of shell regeneration in Helix poniatia concludes on the basis of as yet un published data on carbonic anhydrase inhibition that (p. 36) the enzyme “¿�is necessary for the formation of calcium carbonate crystals in the regenerating shell.†These studies suggest a role of carbonic anhydrase in shell formation but are inconclusive in establishing it. The two inhibitors used by Wilbur and Jodrey, 2-benzothiazolesulfonamide and Diamox, exhibited quite different extents of inhibition of Ca45 deposition in the intact animal, only Diamox affecting deposition in a manner consistent with the hypothesis that the results were due to carbonic anhydrase inhibition. Even though in the mantle-shell preparation used in part of the studies the two drugs had effects consistent with the idea that the enzyme is important in determining the rate of calcium deposit, the side effects of one of the drugs suggest caution in interpretation of the data. Further, the 1 This study has been supported in part by a grant from the Southern Fellowships Fund.
In the gastropod, scaphopod, lamellibranch, and cephalopod gastrulae a thickened portion of the posttrochal region is referred to as the embryonic shell field. It invaginates and gives rise to the shell gland. In species with an at least temporarily external shell, the shell gland evaginates and again forms a shell field. In lamellibranchs, the shell field grows into two halves connected by the ligament-secreting isthmus. In polyplacophorans plate fields are produced without invagination. Slugs and endocochleate cephalopods overgrow the embryonic shell field to form an internal shell sac. The calcified part of the shell is secreted by the flattened central region. The periostracum has its origin in the permanently thickened peripheral region of the shell field. In many forms, this region is depressed in a periostracal groove. If the shell is external, the central region of flattened cells, the mantle roof, along with the two or three marginal folds of the free mantle edge and, in species with internal shell, the shell sac are parts of the mantle. The shell field descends from the first somatoblasts. Either of 2 d or 2 c alone is able to form the shell field. There are arguments that the formation of the embryonic shell field is not autonomic, but induced by the entoderm during a period of contact. The shell gland and the shell field grow by mitotic cell divisions. Cells secreting organic material are highly prismatic, have a well developed ergastoplasm and large dictyosornes, and contain much peroxidase. The secretion of calcium manifests itself in very flat cells, rich in alkaline phosphatase and glycogen. The shell gland and the rosette of ectocochleate conchifera together are homologous to the proximal part of the shell sac in slugs and endocochleate cephalopods.
The combined effects of salinity and temperature on survival and growth of larvae of the mussel Mytilus edulis (L.) were studied. The effects of salinity and temperature are significantly related only as the limits of tolerance of either factor are approached. Survival of larvae at salinities from 15 to 40 is uniformly good (70% or better) at temperatures from 5 to 20C, but is reduced drastically at 25 C, particularly at high (40) and low (20) salinities. Larval growth is rapid at a temperature of 15 C in salinities from 25 to 35, at 20 C in salinities from 20 to 35. Optimum growth occurs at 20 C in salinities from 25 to 30. Growth decreases both at 25 and 10 C; the decline is most drastic at high (40) and low (20) salinities.
X-ray powder diffraction was used to study the calcification of the first larval shell of Ostrea edulis (sampled in Limski kanal, Istria, Adriatic Sea in April 1986) from the trochophore stage to the veliger larvae (prodissoconch I), and development of the latter up to several days postfertilization (prodissoconch II). In the first stage, only the amorphous component is present (periostracum and organic matrix). The beginning of shell formation is manifested by the appearance of calcite (up to 1–4% of the total vol.) and then aragonite (2 to 7%). In a later stage of the veliger larvae the fraction of calcite decreases, as well as the fraction of the amorphous component, while the fraction of aragonite rapidly increases. In the prodissoconch II stage, aragonite is dominant, with a very small amount of amorphous component and traces of calcite. In contrast, the valves of the adult O. edulis are composed mainly of calcite, with traces of aragonite.
Study of the chemical composition of shell of exoskeletonous organisms in the past has required the sacrifice of the organism. Because the beam of the proton microprobe is relatively nondestructive and analyzes the surface layer of the shell, organisms do not have to be killed. The present paper presents results of a preliminary experiment in which distribution of elements (Na to Sr) in shell of living juvenile oysters, Crassostrea virginica (Gmelin), was studied in situ with a proton microprobe at monthly intervals for four months. The relative concentration of 16 elements was measured in the newly deposited prismatic edge of the right valve of three oysters reared in controlled laboratory conditions. Na, Mg, Al, Si, S, Cl, K, Ca, Ti, Cr, Mn, Fe, Cu, Zn, Br, and Sr were detected in concentrations as low as a few parts per million relative to the concentration of standards added to pure CaCO3. Concentration of elements varied nominally among shells of the three individual oysters and in their successive ontogenetic stages. Fluctuations in concentration of Na, Mg, S, Cl, Ca, Mn, Fe, Cu, and Zn were generally similar in the two normally growing oysters, but differed from those in the oyster that stopped growing. Trends in concentration of Al, Si, and Sr were similar in the three oysters: those of Br were variable. Relative concentrations of Na, Cl, S, Mn, Fe, and Zn increased slightly with age of oysters, that of the other elements stayed relatively constant. Concentration of most elements was higher in shell than in seawater. Variable concentrations, especially of Na, Cl, and Si in valve edges, tend to support the hypothesis of earlier workers that separate mineral phases are present as impurities entrapped within the shell during calcification.
X-ray powder diffraction was used to study shell calcifications of the oyster Ostrea edulis, sampled in the Limski Kanal, Istria (Adriatic Sea), in May 1992. All the developmental stages were followed, from the embryonic
stage through the transition between the trochophore and veliger larva (prodissoconch I and II) and later, after swarming,
the pelagic free-swimming larval stages, up to their settlement and attachment (from the D-shaped to the fully formed pediveliger
larva), and finally during metamorphosis and juvenile stages (dissoconch). In the first gastrula stage, only an amorphous
tissue is present (a periostracum and organic matrix). The beginning of shell formation (at the end of gastrulation) in early
trochophores is manifested by the appearance of calcite (up to 1–7% of total volume) and then aragonite (about 1%). In the
later stage of the veliger larva the fraction of calcite decreases as well as the amorphous fraction, while the fraction of
aragonite rapidly increases. In the prodissoconch II stage and during the whole pelagic period aragonite is dominant, accompanied
by a very small amorphous fraction and traces of calcite. The shell mineral composition does not change until metamorphosis,
whereupon the fraction of calcite rapidly increases and the fraction of aragonite decreases. The postmetamorphic valves of
the juvenile and adult oyster consist mainly of calcite, except the resilium and myostracum which remain aragonitic, possibly
as a continuation of the inner layer of the larval shell.
Thesis (M. Sc.)--University of Wales (U.C.N.W., Bangor: Animal Biology), 1986. References: leaf 37.
1. Sarcoplasmic reticulum vesicles and mitochondria were prepared from red and white skeletal muscles of the rabbit. The preparations were characterized in terms of their specific activities of citrate synthase, basal (Mg2+-dependent) and Ca2+-dependent ATPase (the latter two in the presence of NaN3 and ouabain), and their specific carbonic anhydrase activities were determined. 2. Skeletal muscle mitochondria had high specific activities of citrate synthase (700-1200 mu. mg protein-1) and low carbonic anhydrase activities (0.1-0.4 u. ml mg protein-1). The latter are likely to be due to a contamination of the preparations with sarcoplasmic reticulum (s.r.). 3. Preparations of s.r. vesicles showed negligible activities of citrate synthase and the expected differing patterns of basal and Ca2+-dependent ATPase in red and white muscles. Specific carbonic anhydrase activities in s.r. from both muscle types were high (2-4 u. ml mg protein-1). The highest carbonic anhydrase activity, 11 u. ml mg protein-1, was found in s.r. from rabbit m. masseter. The inhibition constant of s.r. carbonic anhydrase towards acetazolamide was 4-6 x 10-8 M and similar but not identical to that of cytosolic carbonic anhydrase II. 4. It appears possible that the carbonic anhydrase II-like enzyme previously found by us in muscle homogenates originates from the s.r. 5. Histochemical studies using the dansylsuphonamide method described previously showed an intracellular pattern of carbonic anhydrase staining compatible with the presence of the enzyme in s.r.: spots homogeneously distributed across the fibre cross-sections in transversely sectioned fibres and thin, longitudinally oriented, bands in longitudinally sectioned fibres. 6. It is estimated that s.r. carbonic anhydrase accelerates CO2 hydration within the s.r. approximately 1000-fold. Thus, CO2 and HCO3- react fast enough to provide a rapid source and sink for protons leaving and entering the s.r. in exchange for Ca2+.
We have studied the effects of carbonic anhydrase inhibition on the hypercapnic ventilatory response of the pulmonate snail, Helix aspersa, in an isolated brain-pneumostome preparation. We found that the cell permeant carbonic anhydrase inhibitor, acetazolamide (ACTZ), increased pneumostomal opening and ventilation during normocapnia (2-3% CO2) and decreased the rate of pneumostomal response to step changes in CO2 (4.5%), but did not change the steady-state ventilatory response to elevated CO2 (4.5%) compared to the inactive ACTZ analogue, N2-substituted 2-acetylamino-1,3,4-thiadiazole (Cl 13850). In contrast, the cell impermeant carbonic anhydrase inhibitor, quartenary ammonium sulfonilamide (QAS), had no effect on the pneumostomal response to CO2 compared to Cl 13850. Using Hansson's histochemical technique to stain for carbonic anhydrase activity, we identified a small number of neurons in the subesophageal ganglia that exhibited carbonic anhydrase activity. Some of these cells were in the region of CO2-sensitivity. In conclusion, carbonic anhydrase inhibition slows the ventilatory response to rapid changes in CO2, but does not affect the intrinsic ability of H. aspersa to respond to CO2. The ventilatory effects of carbonic anhydrase inhibition may be attributed to the intracellular actions of the carbonic anhydrase enzyme.
It is believed that the polymorphism observed in calcium carbonate crystals, such as aragonite and calcite in mollusk shells, is controlled by organic matrix proteins secreted from the mantle epithelia. However, the fine structures of these proteins are still unknown, and to understand the molecular mechanisms of mineralization process, detailed structural analyses of the organic matrix proteins are essential. For this, we have carried out purification, characterization, and cDNA cloning of nacrein, which is a soluble organic matrix protein in the nacreous layer of oyster pearls. Northern blot analysis showed that the nacrein transcript was specifically expressed in mantle pallial. Analysis of the deduced amino acid sequence revealed that the protein contained two functional domains: one was a carbonic anhydrase and another was a Gly-Xaa-Asn (Xaa = Asp, Asn, or Glu) repeat domain; however, the carbonic anhydrase domain was split into two subdomains with insertion of the Gly-Xaa-Asn repeat domain between them. Our findings suggest that nacrein actually functions as a matrix protein whose repeated Gly-Xaa-Asn domain possibly binds calcium and as a carbonic anhydrase that catalyzes the HCO3- formation, thus participating in calcium carbonate crystal formation of the nacreous layer.
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