Calcium localization in the shell-forming tissue of the freshwater snail,Biomphalaria glabrata: a comparative study of various methods for localizing calcium

Summary The routes calcium might take across the mantle to the shell have been investigated with various electron-microscopical techniques in the freshwater snailBiomphalaria glabrata (Planorbidae, Basommatophora).In chemically-fixed tissue, calcium was precipitated with a tannic acid-antimonate technique in predominantly the intercellular spaces of the outer mantle epithelium and the interstitium below it. Some vacuoles of the outer mantle epithelium and one type of mucus cell in the inner mantle epithelium also contained precipitate. The presence of calcium in the precipitates was proved by electron energy loss spectroscopy combined with electron spectroscopic imaging. Incubation with lead acetate and uranyl acetate revealed binding-sites for calcium in the intercellular spaces of the epithelia interstitium and the mucus cells of the inner mantle epithelium. Precipitates were also seen after all incubations in the calcium spherites of the connective tissue.The concentrations of calcium and other elements were analysed in freeze-dried ultrathin sections of cryofixed mantle tissue by means of energy-dispersive X-ray microanalysis. Only in mitochondria of the musculature could high amounts of calcium and phosphorous be detected.     

Electron microscope studies of the intracellular origin and formation of calcifying granules and calcium spherites in the hepatopancreas of the snail,Helix pomatia L.

Summary It has been previously demonstrated that the hepatopancreas of the snail, Helix pomatia, produce proteinaceous particles (termed b-granules), which are involved in the process of calcification. In calcium cells of this organ calcium spherites arise from these granules. The present study retraces the intracellular development of b-granules from Golgi vesicles and from Golgi saccules encircling the cytoplasmic areas up to the formation of calcium spherites. It is suggested that the mature b-granules are well-defined cytoplasmic units with calcifying capacity. The fine structure of b-granules and calcium spherites is described. The possible function of various cell organelles in the formation of the b-granules and spherites is discussed.This investigation was supported by grants from the Swedish Natural Science Research Council. The assistance of Mrs. I. Rehnberg is gratefully acknowledged.     

Role of intestinal mucus in crystal biogenesis: an electron-microscopical,diffraction and X-ray microanalytical study

Summary In the posterior intestine of the sea-water eel, mucus plays an important role in biocrystallization of calcium ions. By means of transmission and scanning electron microscopy associated with X-ray microanalysis and X-ray diffraction it has been possible to determine the role of mucous fibers as nucleation sites. Biocrystallization occurs in 2 steps: (1) Calcification of mucus. As soon as mucus is excreted in the intestinal lumen, it is loaded with calcium, as shown by lanthanum affinity and X-ray microanalysis on freeze-dried tissues. (2) Genesis of crystals. Needleshaped crystallites build up in coalescent spherites in the intestinal lumen near the microvilli. Genesis occurs as follows: (a) crystallite mineralization by nucleation in an organic matrix composed of glycoproteinaceous mucous fibers, followed by the appearance of spherites; (b) coalescence in spherites and association of spherites in rhombohedra; (c) extrusion of organic material during the final step of crystallization.     

Calcification in insects: The dwelling-tube and midgut of machaerotid larvae (Homoptera)

The dwelling-tubes of machaerotid larvae consist of a mineralized organic scaffolding of mucofibrils. The mineral component accounts for 85 per cent of the dry weight and is composed of calcium, ferrous iron, manganese, magnesium, potassium, sodium, phosphate, carbonate, and chloride and of these the major ions are calcium and carbonate. Ferric iron in the form of ferritin is probably also present.Calcium, manganese, magnesium, and phosphate are derived from spherites secreted by a specialized region of the midgut. Calcium and phosphate are present in the spherites, probably as amorphous tricalcium phosphate. Subsequent to secretion the spherites are slowly dissolved and the calcium is incorporated into the dwelling-tube as calcium carbonate. Thus it appears that within the dwelling-tube calcium phosphate is converted to calcium carbonate.Ferritin and ferrous iron are secreted by another specialized region of the midgut and are also incorporated into the dwelling-tube.     

Calcium reabsorption in the posterior caeca of the midgut in a terrestrial crustacean,Orchestia cavimana

Summary During molting, the epithelium of the posterior caeca (PC) of the midgut in the terrestrial crustacean, Orchestia cavimana, is active in calcium turnover. In the preexuvial period, epithelial cells that progressively differentiate into cell-type III secrete ionic calcium (originating from the old cuticle) from the base to the apex of the cell within a typical extracellular network of channels; the calcium is then stored in the PC lumen as calcareous concretions. Immediately after exuviation, the epithelial cells rapidly differentiate into cell-type IV, reabsorbing calcium from the concretions through successive generations of spherites which quickly appear, grow, and then disappear from the apex to the base of the same extracellular network. The PC epithelium is thus alternatively calcium-loaded and unloaded. When the calcium-reabsorbing process is complete (average 48 h after exuviation), the epithelial cells again differentiate into two different regional cellular types (cell-type I in the distal segment and cell-type II in the proximal segment) characteristic of the intermolt period.The dynamic changes in the PC epithelium during the postexuvial period are discussed, including the characteristic features of cell-type IV and of the reabsorption spherites.     

Alterations in the fine structure of cytoplasmic organelles in the hepatopancreatic cells of shell-regenerating snail,Helix pomatia L.

Summary The investigation of hepatopancreatic cells following damage to the shell revealed hypertrophic alterations of several Cytoplasmic organelles. The network of endoplasmic reticulum was extensively developed within both digestive and calcium cells. The arrangement of tubules exhibited a peculiar hexagonal pattern. It is suggested that the reticulum is of agranular type and may be engaged in the transport of lipids and calcium ions. Remarkable alterations were observed in mitochondria, apparently as the result of their hyperfunction. Mitochondria with reduced cristae, with two-layered transverse septum, and with bleb-like protrusions, occurred frequently. In the calcium cells, helically entwined fibres appeared among the fragments of disintegrated calcium spherites. The results of the investigation and the possible presence of collagen in the fibres originating from disintegrated spherites are discussed.This investigation was supported by grants from the Swedish Natural Science Research Council, which are gratefully acknowledged. The author wishes to thank Mrs. I. Rehnberg for technical assistance.     

Mineral congregations, “spherites” in the midgut gland ofCoelotes terrestris (Araneae): Structure,composition and function

Summary Spherites in the digestive and secretory cells of the midgut gland of the agelenid spiderCoelotes terrestris were studied by electron microscopy and histochemical methods. Spherites measured 1–6 m in diameter and were characterized by alternating layers of electron dense and electron lucent material. The main-components of spherites were calcium phosphates and calcium carbonates. Guanine and barium, as well as aminopeptidase and alkaline phosphatase were also present. The matrix consisted of proteins and carbohydrates. Numerous spherites were found together with excretory products within the excretory vacuoles of the digestive cells.Spiders fed with food containing lead, showed deposition of the metall in the spherites. It is then proposed that spherites, aside from their role in storing calcium and other ions, may function in detoxification of heavy metals.     

A study of the concentrically laminated concretions, 'spherites' in the regenerative cells of the midgut of Lepidopterous larvae

Concentrically laminated granules, spherites, are sometimes found in the regenerative cells of midgut of some species of lepidopterous larvae. The spherites are formed in cytoplasmic vesicles just before ecdysis and disappear during the differentiation of the regenerative cells to columnar and goblet cells. They function as intracellular stores of compounds used in the growth of the cell. Phosphates of magnesium and perhaps calcium are probable constituents. Spherites are sometimes also found in the degenerating columnar cells where they are excreted into the lumen with the exfoliating epithelium. The phenomenon of periodic precipitation which is the physical-chemical basis of the formation of spherites is discussed.     

Inorganic polyphosphates are stored in spherites within the midgut of Anticarsia gemmatalis and play a role in copper detoxification

Inorganic polyphosphates (PolyP) are widespread molecules that have been shown to play a role in metal detoxification and heavy-metal tolerance. In the present report, we investigated the functional role of spherites as PolyP-metal binding stores in epithelial cells of the midgut of Anticarsia gemmatalis, a lepidopteran pest of soybean. PolyP stores were detected by DAPI staining and indirect immunohistochemistry as vesicles distributed in columnar cells and around goblet cell cavities. These PolyP vesicles were identified as spherites by their elemental profile in cell lysates that were partially modulated by P- or V-ATPases. PolyP levels along the midgut were detected using a recombinant exopolyphosphatase assay. When copper was added in the diet of larva, copper detection in spherites by X-ray microanalysis correlated with an increase in the relative phosphorous X-ray signal and with an increase in PolyP levels in epithelia cell lysate. Transmission electron microscopy of chemically fixed or cryofixed and freeze substituted tissues confirmed a preferential localization of spherites around the goblet cell cavity. Taken together, these results suggest that spherites store high levels of PolyP that are modulated during metal uptake and detoxification. The similarity between PolyP granules and spherites herein described also suggest that PolyP is one of the main phosphorous source of spherites found in different biological models. This suggests physiological roles played by spherites in the midgut of arthropods and mechanisms involved in heavy metal resistance among different insect genera.     

A morphological and electron-microprobe study of the inorganic composition of the mineralized secretory products of the calciferous gland and chloragogenous tissue of the earthworm,Lumbricus terrestris L.

Summary The two pairs of lobes of the calciferous gland of the earthworm Lumbricus terrestris are specialized oesophageal diverticulae that secrete spherites ranging from 0.5–7.0 m in diameter. Correlative transmission and scanning electron microscopy indicated that the spherites (which are predominantly CaCO₃) are formed extracellularly in distinctive bays bounded by secretory-cell processes, and are mobilized anteriorly from the gland lumina to the lumen of a non-secretory pouch, where the majority coalesce and undergo phase transformation to concretions 0.5–1.0 mm in diameter consisting of a mass of cuboidal crystals with facets up to 40 m. The distribution of Sr (0.1 ml 5 % SrCl₂ injected into the posterior coelomic cavity) was monitored in the mineralized secretory products of the calciferous glands by X-ray microanalysis of 10 m — thick air-dried cryostat sections in a SEM. Strontium was not detected in chloragosomes at 2 h and 24 h post-injection. Strontium was transported anteriorly and specifically incorporated into gland spherites (detectable within 2h). This technique of Sr localization afforded sufficient structural and analytical resolutions to provide a confirmation of the sequence of extracellular changes in the gland/pouch system. In addition we were able to distinguish a population of growing spherites from the vast majority of mature spherites; size alone was a singularly poor indication of spherite growth.The major element constituents of the chloragosomes were P, Ca and Zn (Ca: P ranging from 0.4 to 1.0; Zn: P from about 0.05 to 0.45). Analysis of individual spherites showed that Ca was probably bound to P or P-containing matrix components, whilst Zn was probably linked to one or more different but unknown constituents.     

A histological and ultrastructural study of the cells of the mantle edge of a marine bivalve, Cerastoderma edule

The cells of the mantle edge of Cerastoderma edule are described after light and electron microscopical observations. Histochemical tests for calcium in the mantle edge and digestive gland (Dahl, 1952; McGee-Russell, 1958) and analytical electron microscopy of the mantle edge of C. edule both failed to show calcium. Similar results were obtained for Mytilus edulis and Chlamys opercularis. However, calcium was detected in the digestive gland of the terrestrial gastropod Helix aspersa. The outer secretory fold of the mantle edge is composed of tall columnar cells. These cells have highly convoluted lateral cell membranes with which many mitochondria are closely associated. These features are indicative of an ion pump which could move calcium from the mantle space to the extrapallial cavity (compare with Bubel's findings, 1973b). There are many features of the cells lining the periostracal groove of C. edule that have not been reported previously (e.g. Bubel, 1973b) and which are now discussed. The periostracal sheet arises within a line of basal cells in the fundus of the periostracal groove. Within these cells the periostracum in section has a spiral form. It is suggested that the newly formed periostracum adheres to the microvillous border through secretions produced from the middle fold cells lining the groove. During its passage along the groove the periostracum is gradually thickened by secretions from the outer fold cells.     

Biomineralisation markers during a phase of active growth in Pinctada margaritifera

To search for the biochemical parameters involved in calcium and carbonate transport during crystal formation and biomineralisation in nacreous molluscs, the carbonic anhydrase activity, the levels of calciotropic hormones in hemolymph and in tissues and the circulating concentration of calcium were measured in pearl oysters (Pinctada margaritifera) during a phase of active growth. Activity of carbonic anhydrase in gill tissue increased linearly with age of the animals, while no age variation in activity was noted for the mantle. The circulating level of total calcium increased during the growth of the animals. Calciotropic hormones were radioimmunoassayed in gill, mantle and hemolymph. Only a calcitonin-gene related peptide (CGRP) could be detected and its concentration decreased as a function of growth, both in hemolymph and mantle. No variation in CGRP concentration with age was observed in gill tissue. Our data demonstrate that carbonic anhydrase and a molecule biologically and immunologically related to CGRP are involved during growth of the animals. In addition, this study shows the presence of three main calcium compartments, gill, hemolymph and mantle, involved in the biomineralisation process.     

Observations on the proventricular glands (‘organes racémiformes’) of the oribatid miteChamobates borealis (Acari,Oribatida): an organ of interest for studies on adaptation of animals to acid soils

The proventricular glands of the oribatid miteChamobates borealis (Trägårdh, 1902) were investigated by electron microscopy and histochemistry, and their function was tested in a laboratory experiment. Specimens of the same species collected during a field study also were investigated.The cells of the proventricular glands are characterized by great amounts of mineral spherites and seem to be sensitive to alterations of environmental pH and concentration of CaCO₃. It was demonstrated that a reduction of pH leads to a decrease of the spherites, whereas at a high pH the number of spherites increases.The proventricular glands with their spherites seem to play an important role in regulation of mineral budget, pH and detoxification of heavy metals.     

河蚌培养组织的几种生化成分分析

本文分析测定了三角帆蚌，褶纹冠蚌和背角无齿蚌外套膜培养组织及其培养液中的氨基酸，牛磺酸及钙含量。在珍蛛中含量较高的丙的氨酸和甘氨酸分别增加５４１％和９１％。三种蚌在培养中牛磺酸含量增加５．７８％到３倍，培养组织的钙含量增加１倍左右。同时测定了培养组织的碱性磷酸酶活性，培养组织与河蚌外套膜具有相近的比活及相对酶活。结果表明，河蚌外套膜在离体培养条件下，也具有分泌珍珠质的能力。     

Content,absorption quantities and intracellular storage sites of heavy metals in Diplopoda (Arthropoda)

By means of atomic absorption spectrophotometry, concentrations of more than 2500 mg kg^–1 Pb, 150 mg kg^–1 Zn, and 320 mg kg^–1 Cd could be detected in the intestine tissues of diplopods from a lead and silver smelter's spoil bank. While only small portions of the ingested lead and cadmium are absorbed in the midgut of these diplopods, the zinc uptake into the midgut epithelium reaches 33.8–37.5% of the zinc content in the food pulp when the animals were contaminated acutely. However, after long-term contamination with zinc, absorption and excretion of this metal balanced one another. Absorbed lead and cadmium are predominantly stored in the midgut cells of the diplopods; unspecific precipitation of heavy metal showed the spherites of the resorptive epithelial cells to be the main accumulation sites. Zinc is for the most part localized in or near the cuticle; electron energy loss spectroscopy and ESI electron spectroscopic imaging, however, showed this metal to be present also in the spherites of the midgut's resorption cells. These spherites are assigned to belong to the type A granule group since (i) they are concentrically structured, (ii) they are shown to contain great amounts of calcium and (iii) copper, a class B metal, could not be detected in these deposits.     

Structure of the Malpighian tubule cells and annual changes in the structure and chemical composition of their spherites in the cave cricket Troglophilus neglectus Krauss, 1878 (Rhaphidophoridae,Saltatoria)

Periodical changes in the structure of spherites in the Malpighian tubule cells of the cave cricket Troglophilus neglectus were studied to elucidate their role during the cricket's life cycle in natural circumstances. Special interest was given to the dormant overwintering period when we hypothesized that the primary role of spherites is to supply minerals for basic vital processes. The investigation was carried out by light and transmission electron microscopy, energy dispersive X-ray spectroscopy, electron energy-loss spectroscopy and energy-filtering TEM. Spherites are present only in the middle Malpighian tubule segment, consisting of Type 1 cells, characterized, among other features, by a round, apically placed nucleus and numerous spherites, and a few Type 2 cells with an elongated nucleus in the centre and sparse spherites. At the beginning of dormancy in November juveniles, minerals are accumulated in spherites and then decline until March. In one-year-old May larvae, spherites are commonly rich in minerals, and from July onwards they are progressively exploited in the adults. Spherite destruction starts with apoptosis in senile October individuals. The findings suggest that the mineral supply of spherites in Malpighian tubules is crucial to supporting vital processes throughout the life cycle of T. neglectus.     

Pulse discharge of calcium through a demoralizing epithelium in the crustacean Orchestia: Ultrastructural cytochemistry and X-ray microanalysis

During the post-exuvial period, the posterior caeca of the terrestrial crustacean Orchestia quickly reabsorb the calcareous concretions stored during the pre-exuvial period through successive generations of spherules which appear, grow, then disappear from the apex to the base of a typical network of extracellular channels. The caecal epithelium is thus alternatively calcium-loaded and unloaded. Ultrastructural cytochemistry, using direct precipitating methods (potassium pyroantimonate, oxalic acid and sodium fluoride) or indirect substitution method (lead acetate) reveals that this extracellular pathway could be the main route for a massive transport down a concentration gradient of ionic or ionizable calcium which temporarily precipitates into instable spherules basally solubilized. Quantitative microanalysis of both frozen dry sections and anhydrous thin sections of cryosubstituted resin-embedded material indicates maximal paracellular calcium concentrations during the loading phases, which may be responsible not only for the calcification of the spherules but also for the abnormally high calcium content within the cytoplasm and the mitochondria.     

Heavy meromyosin and subfragment-1 from squid mantle myosin, and Ca-sensitivity of their Mg-ATPases

Heavy meromyosin (HMM) and subfragment-1 (S1) were obtained from squid mantle myosin by tryptic digestion and chymotryptic digestion, respectively. Squid HMM(T) and S1(CT) preparations contained stoichiometric amounts of the two types of light chain subunit; regulatory light chain, LC-2, and essential light chain, LC-1. No difference was detected in the chymotryptic digestibilities of squid mantle myosin in Ca-medium and in EDTA-medium. This is in contrast to the digestibility of scallop adductor myosin. The Mg-ATPase activity of HMM(T) alone and that of acto-HMM(T) were both sensitive to calcium ions. In contrast, the activity of S1(CT) alone and that of acto-S1(CT) were both insensitive to calcium ions. The affinity of HMM(T) for actin was not affected by calcium ions, but the amount of HMM(T) bound to actin was increased by calcium ions from 20% to 60% of the total amount of HMM(T). On the other hand, the actin affinity of S1(CT) and the amount of S1(CT) bound to actin were both unaffected by calcium ions. The role of calcium ions in the regulation of contraction in molluscan muscles is discussed.     

Evidence against calcium as a mediator of mitochondrial dysfunction during apoptosis induced by arachidonic acid and other free fatty acids

Apoptosis is often accompanied by activation of phospholipase A(2), causing release of free fatty acids (FFAs), which in turn are thought to contribute to the loss of mitochondrial transmembrane potential (Deltapsi(m)). In these experiments, we asked whether calcium plays a role as an intermediate in this process. A total of 14 FFAs were compared for their ability to cause loss of Deltapsi(m) and for their ability to affect levels of intracellular calcium. Among the FFAs, unsaturated FFAs tended to induce apoptosis while saturated FFAs did not. Arachidonic acid (AA) was most damaging, causing loss of Deltapsi(m) and cell death in 8-10 h while linoleic acid, gamma-linolenic acid, and docosapentaenoic also strongly induced apoptosis. Effects of the FFAs on levels of intracellular calcium were very different. Many caused strong calcium responses; however, the ability to induce a strong calcium response was not predictive of ability to induce apoptosis, and overall, we did not find a correlation between apoptosis and calcium induction. Also, verapamil and TMB-8 were able to block the calcium response, but these inhibitors did not prevent loss of Deltapsi(m), indicating that the calcium response is not necessary for FFA-induced loss of Deltapsi(m). In contrast, we found that cyclosporine A could inhibit the AA-induced loss of Deltapsi(m) with both whole cells and isolated mitochondria, confirming that the antimitochondrial effects of FFA can stem from direct effects on the mitochondrial permeability transition pore. Finally, we show that the strong apoptosis-inducing activity of AA may stem from its ability to selectively induce its own release.     

Concomitant Localization of Dopa Oxidase and Adenosine Triphosphatase in Cryostat Sections

Cryostat sections fixed 10 min in calcium formalin were incubated sequentially in 0.1% DOPA, 2 hr at 37 C, and ATPase substrate 1% hr at 37 C. The enzymatically produced calcium phosphate was visualized by 0.2% glyoxal-bis(2-hydroxyanil) (GBHA) in alkaline ethanol as a red precipitate. Nonspecific protein-bound calcium ions which obscured active sites in such formalin-fixed material were successfully removed by treatment with 0.5% ethylenediaminetetraacetic acid at pH 7.0 for 1 min before treatment with GBHA. Phosphatase sites were red; DOPA active sites, black. The method was also successfully applied to the demonstration of alkaline phosphatases. Acetone fixation inhibited both enzymes; fixation in 70% alcohol suppressed the DOPA reaction and partially inhibited ATPase.