Calcineurin Plays an Important Role in the Shell Formation of Pearl Oyster (Pinctada fucata)

Calcineurin (CN) is a multifunctional protein involved in many important physiological processes in mammalians, but the function of CN in mollusks is still largely unknown. In the present study, through the shell regeneration system, the changes of enzymatic activity of CN were determined in the process of shell regeneration in pearl oyster Pinctada fucata. CN was activated immediately and continuously in the shell regeneration process. The speed of shell regeneration was measured and the ultrastructure of inner shell surface was observed by scanning electron microscopy after inhibiting CN by intramuscular injection of immunosuppresant cyclosporine A (CsA). The results showed that the speed of shell regeneration was delayed and the morphology of calcite and aragonite in the inner shell surface became abnormal when CN was inhibited by CsA. Meanwhile, RT-PCR analysis revealed that the expression of P. fucata BMP-2 in mantle tissue decreased with CsA injection. In vitro secretion level of proteoglycans (PGs) in primary cultures of mantle cells was also decreased when mantle cells were exposed to CsA. Taken together, our results, for the first time, show that CN is involved in the shell formation through regulating the expression of Pf-BMP-2 in mantle tissue, which controls the secretion of PGs/GAGs of the mantle epithelial cells.     

An ultrastructural study of the mantle of the barnacle, Elminius modestus Darwin in relation to shell formation

The fine structure of the mantle and shell of the barnacle, Elminius modestus Darwin has been examined by electron microscopy. The epithelial cells along the outer face of the mantle differ in size, shape, and organelle complexity according to the different components of the shell they secrete. The shell consists of a non-calcareous basis and calcareous mural and opercular plates which are connected by a flexible opercular hinge. Both the basis and opercular hinge are composed of two main units: an outer cuticulin layer and a lamellate component of well ordered arched fibrils. During the deposition of the latter structures morphological changes in the cells occur which may be correlated with the moulting cycle. Preliminary results show that the calcareous plates are covered by an outer epicuticle, which is bordered by a cuticulin layer; the inner calcareous component, consists of an orderly arrangement of organic matrix envelopes within which crystals may be initiated.

The cells lining the inner surface of the mantle are uniform in appearance with a thin cuticle at their free surface which lines the body cavity. The latter structure of the cuticle and manner of its deposition are similar to those of the basis and opercular hinge. Separating the outer and inner mantle epithelial cells is connective tissue which comprises several differing cell types. The possibilities are discussed of the rôle these cells may play in shell deposition. The modes by which underlying cells secrete the different shell components and the cuticle lining the inner face of the mantle, are also discussed.     

Isolation of crustecdysone (20R-hydroxyecdysone) from a crayfish (Jasus lalandei H. Milne-Edwards)

1. A small amount (2mg.) of crustecdysone, a moulting hormone of crustaceans, was isolated from 1 ton of crayfish waste. 2. The purification procedure used was developed with the aid of crustacean and insect bioassays. 3. CM-Sephadex was found to be superior to Sephadex and very effective for the chromatographic separation of crustecdysone from other non-ionic compounds. The higher efficiency of CM-Sephadex is attributed to the greater number of carboxyl groups available for hydrogen-bonding. 4. Reversed-phase chromatography, with butan-1-ol-cyclohexane mixtures as the stationary phase and water as the flowing phase, proved superior to countercurrent distribution with these solvents for the fractionation of purified extracts. 5. A second moulting hormone, deoxycrustecdysone, and the red-concentrating hormone were obtained in a partially purified form.     

Sex-specific mediation of foraging in the shore crab, Carcinus maenas

Experiments were conducted to investigate the sex-specific differences to feeding responses of the shore crab Carcinus maenas throughout the year. Results demonstrate that female shore crabs exhibit stronger feeding responses than males throughout the year with a significantly reduced feeding response in males during the summer months' reproductive season. We also studied the possible function(s) of the moulting hormone, 20-hydroxyecdysone (Crustecdysone) that has been described as a potential female-produced sex pheromone to initiate male reproductive behaviour in a number of crustaceans. We recently presented evidence that for shore crabs this is not the case and now show that the steroid is instead functioning as a sex-specific feeding deterrent protecting the moulting 'soft' female crabs. Whilst male shore crabs were deterred from prey (Mytilus edulis) and synthetic feeding stimulants glycine and taurine when these feeding stimulants were spiked with crustecdysone, intermoult female crabs were significantly less affected and rarely deterred from feeding. This sex specificity of the moulting hormone, in combination with the female sex pheromone, which has no anti-feeding properties, ensures that male crabs mate with soft-shelled, moulted females rather than engage in cannibalism, such as found frequently in cases when soft-shelled females are exposed to intermoult females.     

Seasonal production of moulting hormone in the barnacle Semibalanus balanoides

An analytical procedure for the quantification of ecdysteroids (crustacean moulting hormone) in barnacles was devised so that minimum sample size could be used. A combination of solvent partitions, Sephadex chromatography, silylation and gas chromatography with electron capture detection was devised, enabling ecdysteroids to be determined down to 20 pg. This was used to determine the amount of moulting hormone in a population of barnacles over a 30 month period. Levels varied from barely detectable in winter months to a maximum value of 1.5 μg kg^− 1 of wet weight of barnacles in September. Polar conjugates of 20-hydroxyecdysone were detected only during the winter months. The number of barnacles moulting at any time corresponded roughly to the titre of hormone present at that time.     

Shell formation and calcium transport in the barnacle Chthamalus fragilis

The mantle epithelium of the barnacle Chthamalus fragilis (Darwin) exhibits several ultrastructural features which may serve to regulate the calcification process. At the basis-mural plate and intermural plate junctions where rapid shell growth occurs, cells are characterized by long apical cytoplasmic projections and large intercellular spaces. These features may increase the functional surface area of the epithelium and enable more rapid deposition of calcium. The cells underlying the general shell surfaces contain numerous electron-dense inclusion bodies and show frequent cellular disintegration near the growing shell interface. Release of the granular contents of these inclusion bodies has been observed in both disintegrating and non-disintegrating cells. X-ray microanalysis revealed significantly higher calcium levels in the inclusion bodies than in the surrounding cytoplasm. This suggests a calcium transport role for these inclusion bodies. Cellular debris produced as a result of the disintegration of the mantle cells near the shell may play some role in the formation of the organic matrix of the shell. The presence of large numbers of mitochondria and well-developed apical microvilli in the cells of the inner mantle epithelium suggest that these cells serve to transport calcium into the mantle from the ambient sea water.     

A light- and electron-microscopic study of enzymes in the embryonic shell-forming tissue of the freshwater snail, Biomphalaria glabrata

Abstract. The mode of formation of the molluscan exoskeleton is still poorly understood, but studies on adult snails indicate that enzymes involved in vertebrate bone formation also participate in mollusc shell formation. The enzymes peroxidase, alkaline phosphatase, and acid phosphatase are expressed in a constant pattern and help to identify the different zones of the adult shell-forming tissue. The present study evaluates whether the expression of these enzymes is also a tool for the identification of the developing zones of the embryonic shell-forming tissue. Thus, we analyzed the temporal and spatial activity of the above-mentioned enzymes and of tartrate-resistant acid phosphatase in the shell forming tissues in Biomphalaria glabrata. Embryos of different age groups and adults were studied; alkaline phosphatase activity was seen in very young embryos in the shell field invagination prior to the secretion of any shell material, while peroxidase activity was present from the start of the periostracum production. Acid phosphatase, found in considerable amounts in yolk granules and albumen cells, appeared in the embryonic shell-forming tissue in relatively few Golgi stacks. Tartrate-resistant phosphatase was not present in embryos, but was found in adults in the same zone of the mantle edge as acid phosphatase. Using the enzymes as cell markers, the differentiation of the embryonic shell-forming tissue to the different zones of the adult mantle edge could clearly be followed.     

Experiments on orientation of the intertidal barnacle Tesseropora rosea (Krauss)

The intertidal barnacle Tesseropora rosea (Krauss) exhibits very marked directionality of orientation on sloping and vertical surfaces. The barnacles are orientated so that their cirral fans face the water current; this pattern was found within 1 wk after settlement. Adults also showed similar orientation to the water current at each site.Three manipulative experiments were done in the field to determine the consequences of incorrect orientation by the barnacles. Correct orientation was necessary for T. rosea to maintain its tissue weight, but experimental rotation of barnacles through 90° or 180° made no difference to rates of shell growth, mortality or weight of egg masses. Furthermore, this species was unable to re-orientate once it had metamorphosed, and orientation of newly-settled barnacles was not influenced by that of adults.     

Carbonic anhydrase activity and biomineralization process in embryos, larvae and adult blue mussels Mytilus edulis L.

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. Received: 28 November 1998 / Received in revised form: 30 August 1999 / Accepted: 31 August 1999     

Amorphous Calcium Carbonate Precipitation by Cellular Biomineralization in Mantle Cell Cultures of Pinctada fucata

The growth of molluscan shell crystals is generally thought to be initiated from the extrapallial fluid by matrix proteins, however, the cellular mechanisms of shell formation pathway remain unknown. Here, we first report amorphous calcium carbonate (ACC) precipitation by cellular biomineralization in primary mantle cell cultures of Pinctada fucata. Through real-time PCR and western blot analyses, we demonstrate that mantle cells retain the ability to synthesize and secrete ACCBP, Pif80 and nacrein in vitro. In addition, the cells also maintained high levels of alkaline phosphatase and carbonic anhydrase activity, enzymes responsible for shell formation. On the basis of polarized light microscopy and scanning electron microscopy, we observed intracellular crystals production by mantle cells in vitro. Fourier transform infrared spectroscopy and X-ray diffraction analyses revealed the crystals to be ACC, and de novo biomineralization was confirmed by following the incorporation of Sr into calcium carbonate. Our results demonstrate the ability of mantle cells to perform fundamental biomineralization processes via amorphous calcium carbonate, and these cells may be directly involved in pearl oyster shell formation.     

Irregular calcareous sheets preserved in a conch of the Cretaceous ammonoid Hypophylloceras from Hokkaido,Japan

The mantle tissue is essential for understanding the diverse ecology and shell morphology of ammonoid cephalopods. Here, we report on irregular calcareous sheets in a well-preserved shell of a Late Cretaceous phylloceratid ammonoid Hypophylloceras subramosum from Hokkaido, Japan, and their significance for repairing the conch through the mantle inside the body chamber. The sheets are composed of nacreous layers arranged parallel to the irregularly distorted outer whorl surface. The nacreous sheets formed earlier are unevenly distributed and attached to the outer shell wall locally, whereas the last formed sheet covers a wide area of the outer shell wall. The absence of any interruption of ribbing around the irregular area suggests that these sheets were secreted inside the body chamber from the inner mantle. Gross morphological and X-ray computed tomography observations revealed that the spacing of septal formation was not affected by this event. The complex structure of the irregular sheets suggests a highly flexible mantle inside the body chamber.     

Correlation between the morpho-cytohistochemistry of the outer mantle epithelium of Anodonta cygnea with seasonal variations and following pollutant exposure

Seasonal and experimental conditions induce morphological and cytochemical variations in the outer mantle epithelium (OME) of the freshwater bivalve Anodonta cygnea, probably influencing the shell calcification mechanism. In this study, OME samples were taken from untreated animals in autumn, winter, spring and summer as well as from animals exposed to divalent metals (cadmium, chromium, lead, copper and zinc) and pesticides (setoxidim and dimethoate) and observed by light microscopy. The present results showed that OME cells have larger cell volumes and increased amounts of secreted macromolecules during spring and summer than in autumn and winter. This correlates with higher shell calcification rates in spring and summer and lower shell calcification rates in autumn and winter. The experiments showed that incubation with pollutants for 8 months dramatically reduced the cellular volume so that the density of cytoplasmic material appeared higher that in the control samples. The pronounced changes in OME cells suggest a significant decrease in secretory activity following exposure to toxic agents and this has implications for the shell calcification process.     

Proteomic analysis of the organic matrix of the abalone <Emphasis Type="Italic">Haliotis asinina</Emphasis> calcified shell

Background  

The formation of the molluscan shell 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, most of its protein components remain uncharacterised.     

Neuroendocrine Control Mechanisms in Shell Formation

The brain of Helisoma duryi contains several neurodendocrinecentres. Factors) present in the cerebral ganglia are thoughtto be involved in normal shell growth while neurosecretory substancespresent in the visceral ganglion are involved in the repairof damaged shell. In Lymnaea stagnalis a growth hormone is producedby the cerebral ganglion which stimulates periostracum formationand the calcification of the inner shell layer. The second effectis thought to occur through the action of a mantle edge calciumbinding protein. In Helisoma, mantle collar is able to produce the periostracumin vitro. The presence of brain from a fast growing donor increasesthe amount of periostracum produced by a mantle collar froma slow growing animal. This effect is further enhanced by theremoval of the lateral lobes. The periostracum produced by fastgrowing animals has a higher glycine content than that producedby slow growing snails. The presence of dorsal epithelial tissueenhances the incorporation of calcium into periostracum formedin vitro. These findings suggest that a single factor is present in thebrain of fast growing Helisoma which modulates shell formationrates in vivo and periostracum formation in vitro.     

Correlation between the morpho-cytohistochemistry of the outer mantle epithelium of Anodonta cygnea with seasonal variations and following pollutant exposure

Seasonal and experimental conditions induce morphological and cytochemical variations in the outer mantle epithelium (OME) of the freshwater bivalve Anodonta cygnea, probably influencing the shell calcification mechanism. In this study, OME samples were taken from untreated animals in autumn, winter, spring and summer as well as from animals exposed to divalent metals (cadmium, chromium, lead, copper and zinc) and pesticides (setoxidim and dimethoate) and observed by light microscopy. The present results showed that OME cells have larger cell volumes and increased amounts of secreted macromolecules during spring and summer than in autumn and winter. This correlates with higher shell calcification rates in spring and summer and lower shell calcification rates in autumn and winter. The experiments showed that incubation with pollutants for 8 months dramatically reduced the cellular volume so that the density of cytoplasmic material appeared higher that in the control samples. The pronounced changes in OME cells suggest a significant decrease in secretory activity following exposure to toxic agents and this has implications for the shell calcification process.     

The intracellular distribution of calcium in the mucosa of the avian shell gland

The intracellular distribution of calcium has been studied in the mucosa of the avian shell gland, a tissue which transports large quantities of calcium during discrete time intervals. Ca⁴⁵ was administered to hens either in a single dose followed by sacrifice 5 min later or in repeated doses over an extended period followed by sacrifice 2 hr or 24 hr after the last injection. Subcellular fractions were isolated by differential centrifugation and analyzed for Ca⁴⁵. The Ca⁴⁵ was located principally in the particulate fractions; the concentration (CPM Ca⁴⁵/mg N) was highest in the mitochondrial fraction. Comparisons of (1) the Ca⁴⁵ distribution in shell gland cells with that of liver cells, (2) the alterations which occur due to the phase of the egg laying cycle, (3) the effects due to the time elapsed since the last injection of Ca⁴⁵, and (4) the Ca⁴⁵ distribution of the short term experiments with that of the long term experiments revealed that the mitochondrial fraction of the shell gland appeared to be active in the movement of calcium. The microsomal fraction showed increased values in CPM Ca⁴⁵/mg N when calcification was occurring, which may indicate that the subcellular components of this fraction have a role in calcium transport. The nuclear and supernatant fractions did not seem to be involved in the transport process. The implications of these results concerning the manner by which calcium may be controlled on a cellular level in this system are discussed.     

Untersuchungen zur Häutungsphysiologie der dekapoden Krebse am Beispiel der StrandkrabbeCarcinus maenas

Under constant laboratory conditions, juvenile shore crabs moult at fixed intervals which depend upon their body size. During one moult every crab exhibits increases of the same relative amounts, independent of its absolute size. Basing on the predictable duration of the intermoult period, the morphological changes in the structure of the cuticle and the development of limb-buds, the intermoult period could be divided into 21 different stages. After studying the moulting rhythm in constant milieu, the influence of the following exogenous and endogenous factors upon the moulting rhythm and growth of normal and of eye-stalkless individuals was investigated: temperature, photoperiod, loss of pereiopods, feeding, and presence of larger specimens. From these investigations it became evident that the moulting rhythm is regulated by growth. The crabs are able to moult only after achieving a minimum of tissue growth. So long as this minimum growth is not achieved, a moult-inhibiting hormone is secreted and moulting is prevented. If the moult-inhibiting hormone is absent, moulting hormone is secreted and initiates a moult. Under dangerous conditions, the crabs are able to delay the next moult. Under unfavourable conditions they consume less food than normal. Therefore, the amount of tissue growth which is the necessary prerequisite for moulting is delayed, and continued release of moult-inhibiting hormone prevents the moult. Under conditions favourable for moulting, or demanding moult (e. g. after loss of many pereiopods) the crabs accelerate the moult. Temperature influences the moulting rhythm by indirect effects on the metabolic rate. During further investigations, the variation of the following parameters were determined quantitatively: content of moulting hormone in whole crabs; content of aminoacids, protein, glucose, Na⁺, K⁺, Mg⁺⁺ and Ca⁺⁺ in the hemolymph; pH and osmotic pressure in the hemolymph; and Ca⁺⁺ content in skeleton and whole crabs. All parameters mentioned — excepting pH and K⁺ content of the hemolymph — vary characteristically during the intermoult period. The titre of moulting hormone has 4 different maxima. Of all parameters, only the content of animoacids and protein in the hemolymph vary in the same way as the titre of the hormone. From these results the following conclusions are drawn: The moulting hormone not only initiates the moulting process, but controls it at several stages. Only protein metabolism seems to be under direct control of the moulting hormone which stimulates protein-synthesis. Chitin formation, regeneration, apolysis and ecdysis are indirectly controlled by the moulting hormone through protein metabolism. As in most of the other processes mentioned, the calcification of the new cuticle is not under the direct influence of the moulting hormone. The conclusion ofDigby (1966) that calcification in crabs is an electrochemical process, is confirmed.     

The carbonic anhydrase domain protein nacrein is expressed in the epithelial cells of the mantle and acts as a negative regulator in calcification in the mollusc Pinctada fucata

Signals and organic matrix proteins secreted from the mantle are critical for the development of shells in molluscs. Nacrein, which is composed of a carbonic anhydrase domain and a Gly-X-Asn repeat domain, is one of the organic matrix proteins that accumulates in shells. In situ hybridization revealed that nacrein was expressed in the outer epithelial cells of the mantle of the pearl oyster Pinctada fucata. The recombinant nacrein protein inhibited the precipitation of calcium carbonate from a saturated solution containing CaCl2 and NaHCO3, indicating that it can act as a negative regulator for calcification in the shells of molluscs. Because deletion of the Gly-X-Asn repeat domain of nacrein had a significant effect on the ability of nacrein to inhibit the precipitation of calcium carbonate, it is conceivable that the repeat domain has a primary role in the inhibitory function of nacrein in shell formation. Together these studies suggest that nacrein functions as a negative regulator in calcification in the extrapallial space between the shell and the mantle by inhibiting the precipitation of CaCO3.     

Barnacle larval destination: piloting possibilities by bacteria and lectin interaction

Modulation of metamorphosis in barnacles in response to cues of biological origin is established. The bacteria associated with the barnacles also have a role in such modulations. We isolated the bacteria, Pseudomonas aeruginosa, Bacillus pumilus and Citrobacter freundii from the shell surface of Balanus amphitrite and assayed against its cypris larvae. The former species was promotory while the latter two inhibited cyprid metamorphosis. P. aeruginosa however, when tagged with lectins specific to glucose and its derivatives, mannose and fructofuranose negated the promotory effect. Whereas, tagging of galactose derivatives translated the inhibitory effect of B. pumilus and C. freundii into a promotory one showing that lectins can alter the signals in either direction. Galactose-binding lectins have been identified in the haemolymph of barnacles, which could find their way through the excretory system to the surface. The presence of such lectins could probably provide this organism with an ability to alter the signals or cues. Microscale patchiness of bacteria is also evident on surfaces in the sea. The availability of conflicting cues in patches may help pilot the larvae to their settlement destination. Understanding these controlling mechanisms and interfering with the pathways that are involved in lectin synthesis would be a step forward in antifouling technology.     

Global patterns of gene regulation associated with the development of ectomycorrhiza between birch (Betula pendula Roth.) and Paxillus involutus (Batsch) Fr

The formation of ectomycorrhizal (ECM) root tissue is characterized by distinct morphological and developmental stages, such as preinfection and adhesion, mantle, and Hartig net formation. The global pattern of gene expression during these stages in the birch (Betula pendula)-Paxillus involutus ECM association was analyzed using cDNA microarrays. In comparison with nonsymbiotic conditions, 251 fungal (from a total of 1,075) and 138 plant (1,074 in total) genes were found to be differentially regulated during the ECM development. For instance, during mantle and Hartig net development, there were several plant genes upregulated that are normally involved in defense responses during pathogenic fungal challenges. These responses were, at later stages of ECM development, found to be repressed. Other birch genes that showed differential regulation involved several homologs that usually are implicated in water permeability (aquaporins) and water stress tolerance (dehydrins). Among fungal genes differentially upregulated during stages of mantle and Hartig net formation were homologs putatively involved in mitochondrial respiration. In fully developed ECM tissue, there was an upregulation of fungal genes related to protein synthesis and the cytoskeleton assembly machinery. This study highlights complex molecular interactions between two symbionts during the development of an ECM association.