THE LOVE DART OF HELIX ASPERSA MULLER IS NOT A GIFT OF CALCIUM

The phenomenon of dart shooting in several species of land snailshas still not been explained. We were interested in whetherthe dart can function as a nuptial gift of calcium, as previouslyproposed. Donating calcium would increase the fitness of theoffspring and thereby result in a higher reproductive successfor the donor. We confirmed in Helix aspersa that the developingembryo takes up calcium from the egg shell for the formationof its embryonic shell. However, other results from behaviouralobservations and calcium measurements in various reproductivestructures do not support the calcium hypothesis. We found thatthe dart penetrated the skin in 91.7% or the shootings, butit was internalized by the recipient in only 6.3% of the shootings.The amount of calcium in one dart is roughly equal to that ofone egg, and thus it would not contribute significantly to anaverage clutch of 59 eggs. The spermatophore contains virtuallyno calcium, and therefore it is unlikely that the dart signalsa donation of calcium with the sperm. The dart is also unlikelyto influence egg laying since dart shooting does not predicteither the latency or the productivity of egg laying in theshooter or the recipient. We conclude that the love dart ofHelix aspersa is not a gift of calcium. Instead, we suggestthat it is a vehicle to introduce a substance into the partnerto influence the fate of the donated sperm. (Received 8 November 1996; accepted 25 April 1997)     

Dart formation in Helix aspersa (Mollusca,Gastropoda)

Summary Dart formation in Helix aspersa has been investigated by SEM of isolated darts at progressive stages in their development, and by histology of dart sacs at the same times. Dart formation begins at the tip of a tubercle where a small group of epithelial cells secrete an organic material filling a small CaCO₃ cone that is the first mineralized part of the shaft. Subsequent secretory activity by an increasing area of the tubercle epithelium results in an increase in the diameter and anterior lengthening of the shaft. Continued secretion by the tubercle and dart sac epithelium produces the flare and finally the corona. A pattern of deposition is also evident in the fine structure of the mineral. In the shaft and vanes there is an inner layer of spherulitic prismatic structure which is covered by a layer of irregular patches of simple prismatic structure. The outermost layer of the shaft and vanes has a continuous simple prismatic structure. Two layers are present in the flare, an inner granular amorphous layer and an outer spherulitic prismatic layer. The corona consists of a single rarefied prismatic layer. A mechanism of dart formation is suggested that involves two types of organic matrix, calcifying and non-calcifying. Measurements of the calcium content of darts, dart sacs, and collars indicate that the hemolymph is the probable source of calcium for the dart.     

A new approach of pellet formation of a filamentous fungus -Rhizopus oryzae

The effects of inoculum and medium composition (i.e. potato dextrose broth as carbon source, soybean peptone, calcium carbonate, and metal ions) on pellet formation of Rhizopus oryzae ATCC 20344 have been studied. Metal ions were found to have a significant negative effect on pellet formation while soybean peptone had a positive effect. In addition, potato dextrose broth and calcium carbonate were beneficial to R. oryzae for growing small, smooth pellets during the culture. The study also demonstrated that an inoculum size of less than 1.5x10(9)spores/L had no significant influence on pellet formation although it had large impacts on pellet growth. Thus, a new approach to form pellets has been developed using only potato dextrose broth, soybean peptone, and calcium carbonate allowing for pellet size to be controlled by adjusting inoculum size and the concentrations of potato dextrose broth, soybean peptone, and calcium carbonate in the medium.     

The skeletal organic matrix from Mediterranean coral Balanophyllia europaea influences calcium carbonate precipitation

Scleractinian coral skeletons are made mainly of calcium carbonate in the form of aragonite. The mineral deposition occurs in a biological confined environment, but it is still a theme of discussion to what extent the calcification occurs under biological or environmental control. Hence, the shape, size and organization of skeletal crystals from the cellular level through the colony architecture, were attributed to factors as diverse as mineral supersaturation levels and organic mediation of crystal growth. The skeleton contains an intra-skeletal organic matrix (OM) of which only the water soluble component was chemically and physically characterized. In this work that OM from the skeleton of the Balanophyllia europaea, a solitary scleractinian coral endemic to the Mediterranean Sea, is studied in vitro with the aim of understanding its role in the mineralization of calcium carbonate. Mineralization of calcium carbonate was conducted by overgrowth experiments on coral skeleton and in calcium chloride solutions containing different ratios of water soluble and/or insoluble OM and of magnesium ions. The precipitates were characterized by diffractometric, spectroscopic and microscopic techniques. The results showed that both soluble and insoluble OM components influence calcium carbonate precipitation and that the effect is enhanced by their co-presence. The role of magnesium ions is also affected by the presence of the OM components. Thus, in vitro, OM influences calcium carbonate crystal morphology, aggregation and polymorphism as a function of its composition and of the content of magnesium ions in the precipitation media. This research, although does not resolve the controversy between environmental or biological control on the deposition of calcium carbonate in corals, sheds a light on the role of OM, which appears mediated by the presence of magnesium ions.     

Alginate modified nanostructured calcium carbonate with enhanced delivery efficiency for gene and drug delivery

To improve the performance of nanostructured calcium carbonate in gene delivery, a hydrophilic polysaccharide, alginate, was added to calcium carbonate co-precipitation systems to form alginate/CaCO(3)/DNA nanoparticles. The size and ζ-potential of the nanoparticles were measured by a zetasizer. Due to the existence of alginate chains which retarded the growth of calcium carbonate based co-precipitates, the alginate/CaCO(3)/DNA nanoparticles exhibited a decreased size and enhanced stability in the aqueous solution. To evaluate the gene and drug co-delivery ability, doxorubicin hydrochloride (DOX), a water-soluble anticancer drug, was loaded in the nanoparticles to form alginate/CaCO(3)/DNA/DOX nanoparticles. The in vitro gene transfections mediated by different nanoparticles in 293 T cells and HeLa cells were carried out, using pGL3-Luc as a reporter plasmid. With an appropriate amount of alginate, the gene transfection efficiency of alginate modified nanoparticles could be significantly enhanced as compared with the nanoparticles without alginate modification for the gene delivery systems, as well as the gene and drug co-delivery systems. The study on in vitro cell inhibition effects showed that the cell viability decreased with increasing DOX amount loaded in alginate/CaCO(3)/DNA/DOX nanoparticles. The alginate modification is a useful strategy to improve the calcium carbonate co-precipitation technique for the preparation of gene and drug delivery systems, and the nanoparticles prepared in this study have promising applications in gene and drug delivery.     

A new method for the concentration of Cryptosporidium oocysts from water

A novel method for the concentration of Cryptosporidium oocysts from water has been developed, based upon the precipitation of calcium carbonate. A 10 1 water sample is treated by adding solutions of calcium chloride and sodium bicarbonate and raising the pH value to 10 with sodium hydroxide. Crystals of calcium carbonate form and enmesh particles in the Cryptosporidium oocyst size range. The crystals are allowed to settle, the supernatant fluid is discarded and the calcium carbonate precipitate dissolved in sulphamic acid. The sample can be concentrated further by centrifugation. Recoveries of oocysts from seeded samples of deionized, tap and river water were in excess of 68%.     

Calcium carbonate precipitation in cyanobacterial mats from sandy tidal flats of the North Sea

Precipitated calcium carbonate was found in annual cyanobacterial mats developing on the beaches of the North Sea barrier island Schiermonnikoog (the Netherlands). A variety of different calcium carbonate morphs were found in the cyanobacterial mucous secretions and identified by light- and scanning electron microscopy and X-ray diffraction. Most of the calcium carbonate seemed to be associated with degrading extracellular polymer. It is conceived that supersaturation of calcium carbonate resulted from the periodic evaporation of the mats and from the release of calcium from the cyanobacterial mucous as a result of its degradation. The analysis of the carbon stable isotopic composition of the calcium carbonate showed only a slight depletion of ¹³C, indicating that it did not in major part originated from the decomposition of organic matter. The δ¹⁸O values of the calcium carbonate confirmed the temperature differences between spring and summer but excluded the possibility that excessive evaporative events controlled precipitation. The precipitation of calcium carbonate could represent a potential factor enhancing the stabilization of intertidal siliciclastic sediments through cementing the sand. The discovery of massive calcium carbonate precipitation in these cyanobacterial mats was unexpected and hitherto unknown.     

QUANTIFYING GROWTH AND CALCIUM CARBONATE DEPOSITION OF CALLIARTHRON CHEILOSPORIOIDES (CORALLINALES,RHODOPHYTA) IN THE FIELD USING A PERSISTENT VITAL STAIN1

Growth and calcium carbonate deposition rates of the coralline alga Calliarthron cheilosporioides Manza were quantified by monitoring fronds in the intertidal zone that had been chemically labeled with the nontoxic fluorescent brightener Calcofluor white. This vital stain effectively labeled apical meristems of coralline thalli in the field: fronds exposed for only 5 min had detectable chemical marks at least 1.5 years later. By distinguishing portions of thalli that developed before and after exposure, this methodology permitted accurate measurement of growth and calcium carbonate deposition at each meristem. In Calliarthron, meristematic activity declined with increasing frond size. However, because growing fronds dichotomize, the total number of meristems and the deposition rate of new calcified tissue both increased with frond size. Growth rates reported here suggest that large fronds may not be as old as previously estimated. The Calcofluor white method may improve demographic studies of corallines by resolving growth and age of fronds in the field and may facilitate studies of climate change on calcium carbonate deposition in these ecologically important, calcifying algae.     

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.     

High level of sperm competition may increase transfer of accessory gland products carried by the love dart of land snails

Postcopulatory adaptations that increase reproductive success compared to rivals, like the transfer of accessory gland products that promote paternity, are common when sperm competition occurs among males. In land snails, the dart shooting behavior and its adaptive significance, in promoting individual fitness through enhanced paternity of the successful dart shooter, have been considered such an adaptation. The fitness result gained is mediated by the transfer of mucus components on the love dart capable of altering the physiology of the receiver's reproductive tract. In this context, dart shooting and mucus transfer could be considered as processes targeted by sexual selection. While the effect of dart mucus is beneficial for the dart user, so far it has remained unknown whether its transport is greater when snails experience a higher level of sperm competition. Here, we report results of a study on inter‐ and intraspecific variations of dart and mucus gland morphometry, considered to be traits reflecting the ability of snails to adjust the production and transfer of mucus under varying sperm competition scenarios. We investigated four populations with different densities from four dart‐bearing species, Arianta arbustorum, Cepaea nemoralis, Cornu aspersum, and Helix lucorum. The results indicate that different adaptations of these traits occur among the studied species that all seem to achieve the same goal of transferring more mucus when sperm competition is higher. For example, the presence of longer and more branched mucous glands or an increase in dart surface most likely reflect increased mucus production and enhanced ability of mucus transport, respectively. Interestingly, the species for which the use of the dart is reported to be facultative, A. arbustorum, did not show any variation among the examined traits. To conclude, sexual selection in the form of sperm competition intensity seems to be an important selective force for these simultaneously hermaphroditic dart‐bearing snails, driving differences in sexual traits.     

长期施肥对砂壤质石灰性潮土土壤酸碱缓冲体系的影响

通过长期施肥定位试验,分析了石灰性潮土连续30年施肥处理下土壤pH、碳酸钙和活性碳酸钙含量以及土壤酸碱缓冲容量的变化.结果表明:连续30年施肥处理导致石灰性潮土0 ～20 cm耕层土壤酸化加速,各施肥处理的耕层土壤pH降低0.41 ～0.70;各施肥处理的耕层土壤酸碱缓冲容量为15.82 ～ 21.96 cmol·kg-1,单施氮肥明显降低了土壤酸碱缓冲容量,而增施有机肥可显著提高土壤酸碱缓冲容量.土壤酸碱缓冲容量与土壤碳酸钙和活性碳酸钙含量呈显著正相关,与土壤有机质含量和阳离子交换量的相关性不显著,长期不同施肥处理下石灰性潮土仍处于碳酸钙初级缓冲体系,盐基离子和有机质对土壤酸碱缓冲体系的影响较小.0 ～40 cm土层的土壤碳酸钙和活性碳酸钙含量均高于40 ～ 80 cm土层.与土壤碳酸钙相比,土壤活性碳酸钙更能敏感反映土壤基本理化性状的变化,碳酸钙缓冲体系还可以进一步细分为土壤活性碳酸钙缓冲体系.     

Effects of adding calcium and sodium salts to field soils on the incidence of clubroot

The effect on clubroot of the addition to a mineral soil of calcium carbonate, calcium sulphate and sodium carbonate was studied in two field experiments during a 3-yr period. Applications of calcium carbonate (ground limestone) at 10 or 20 t/ha increased the soil pH from 6×7 to 7×9; sodium carbonate at 7×5 t/ha to pH 8×3 and calcium sulphate (gypsum) at 20 t/ha caused a slight depression in pH to 6×6. 98% of cabbage plants showed clubroot symptoms in the untreated plots after 3 yr and the percentages were 0×7, 1×6 and 66×6 for the calcium carbonate, sodium carbonate and calcium sulphate treatments respectively. Yields were significantly increased by all three materials. There were no significant differences in disease incidence or in yield when calcium carbonate was used at 10 or 20 t/ha either applied as a single application or as two half-rate applications. Soil from the calcium carbonate treated plots was used in a greenhouse experiment where the addition of inoculum of Plasmodiophora brassicae resulted in a large increase in clubroot incidence. There was no evidence that a biological suppressor was present in the high pH, low disease incidence soils.     

Mechanism of calcium accumulation in acetate-fed aerobic granule

High calcium content has been widely reported in acetate-fed aerobic granules, but the reason behind this is unclear yet. By SEM–energy dispersive X-ray mapping analysis, this study showed that the majority of calcium was presented in the central part of the acetate-fed aerobic granule, and the granule shell part was nearly calcium-free. The elemental analysis of calcium ions coupled with the chemical titration of carbonate further revealed that the calcium ions that accumulated in the acetate-fed aerobic granule mainly existed in the form of calcium carbonate (CaCO₃). The formation of the CaCO₃ appeared to be highly dependent on the size of the aerobic granule, i.e., the CaCO₃ precipitation was found only in aerobic granules with radiuses larger than 0.5 mm. These experimental observations with regard to the formation of CaCO₃ in the acetate-fed aerobic granule were further confirmed by the model simulation, which was based on the principles of mass diffusion and carbonate dissociation in liquid phase. This study for the first time showed that the size of the acetate-fed aerobic granule would indeed play an essential role in the CaCO₃ formation, and provided experimental evidence that a crystal CaCO₃ core was not necessarily required for granulation.     

A Syringe-Like Love Dart Injects Male Accessory Gland Products in a Tropical Hermaphrodite

Sexual conflict shapes the evolution of many behaviours and processes involved in reproduction. Nearly all evidence supporting this comes from species where the sexes are separated. However, a substantial proportion of animals and most plants are hermaphroditic, and theoretical work predicts that sexual conflict plays an important role even when the sexes are joined within one individual. This seems to have resulted in bizarre mating systems, sophisticated sperm packaging and complex reproductive morphologies. By far the best-known example of such a strategy in hermaphrodites is the shooting of so-called love-darts in land snails. All known love darts carry a gland product on their outside and enter this into the partner’s hemolymph by stabbing. Here, we show that species of the snail genus Everettia possess a syringe-like dart that serves as a real injection needle. Their dart is round in cross-section, contains numerous channels, and has perforations along its side. Histology and electron microscopy show that these holes connect to the channels inside the dart and run all the way up to the elaborate mucus glands that are attached to the dart sac. This is the first report on a love dart that is used as a syringe to directly inject the gland product into the partner’s hemolymph. Although the exact use and function of this dart remains to be demonstrated, this clearly adds to the complexity of the evolution of reproductive strategies in hermaphrodites in general. Moreover, the perforations on the outside of the love dart resemble features of other injection devices, thus uncovering common design and repeated evolution of such features in animals.     

An Unaccounted Fraction of Marine Biogenic CaCO3 Particles

Biogenic production and sedimentation of calcium carbonate in the ocean, referred to as the carbonate pump, has profound implications for the ocean carbon cycle, and relate both to global climate, ocean acidification and the geological past. In marine pelagic environments coccolithophores, foraminifera and pteropods have been considered the main calcifying organisms. Here, we document the presence of an abundant, previously unaccounted fraction of marine calcium carbonate particles in seawater, presumably formed by bacteria or in relation to extracellular polymeric substances. The particles occur in a variety of different morphologies, in a size range from <1 to >100 µm, and in a typical concentration of 10⁴–10⁵ particles L⁻¹ (size range counted 1–100 µm). Quantitative estimates of annual averages suggests that the pure calcium particles we counted in the 1–100 µm size range account for 2–4 times more CaCO₃ than the dominating coccolithophoride Emiliania huxleyi and for 21% of the total concentration of particulate calcium. Due to their high density, we hypothesize that the particles sediment rapidly, and therefore contribute significantly to the export of carbon and alkalinity from surface waters. The biological and environmental factors affecting the formation of these particles and possible impact of this process on global atmospheric CO₂ remains to be investigated.     

不同培养条件对胶质芽孢杆菌诱导碳酸钙晶体形成的影响

【目的】研究不同培养条件对胶质芽孢杆菌(Bacillus mucilaginosus)菌体形态、数量和分泌的碳酸酐酶(CA酶)活性的影响,以及不同方式培养的菌体与碳酸钙晶体的生长及其形貌、数量之间的联系。【方法】分别采用无氮和有氮培养基培养胶质芽孢杆菌,进行菌体形态、数量及CA酶活性的比较,收集不同培养方式的菌体加入碳酸钙结晶体系中以研究细菌与碳酸钙晶体形成的联系。【结果】在无氮培养条件下,胶质芽孢杆菌数量少、荚膜肥厚,细菌培养液CA酶活力较低;有氮培养条件下,菌体数量多、荚膜单薄,细菌培养液CA酶活力较高。在碳酸钙结晶体系中加入无氮培养的菌体,生成的碳酸钙晶体表面光滑,体积较大但数量较小,加入有氮条件下培养的菌体形成的碳酸钙晶体表面粗糙,数量大但体积较小。【结论】不同培养条件能够引起胶质芽孢杆菌菌体数量、荚膜多糖及CA酶活的明显差异,进而对碳酸钙晶体的生成和形貌产生影响。     

The snail's love-dart delivers mucus to increase paternity

Many of the seemingly bizarre animal behaviours can be understood only by acknowledging the power of sex to shape evolution. A case in point is the so-called love-dart that some terrestrial molluscs shoot at their prospective sexual partners. Given that the likelihood of copulation is not different after solid hits than after complete misses, why do these suitors act so violently towards their chosen mates? Previously, it was shown that successful dart shooting enhances paternity. We conducted an experiment to determine whether the dart achieves its effect by a purely mechanical action or by transferring a bioactive substance. We found that injections of mucus from a gland associated with the dart more than doubled paternity relative to injections of saline. These results support the hypothesis that the dart transfers a substance capable of reconfiguring the spermatophore-receiving organs. While dart shooting probably evolved as the result of sperm competition, a role for cryptic female choice cannot be excluded. Our results imply that if cryptic female choice is operating in this system, it is likely to be based on the properties of the mucus and not on properties of the dart itself. Since we also found evidence of early-male sperm precedence, we conclude that snails can optimize their reproductive success by mating with virgins and shooting their darts accurately.     

厚壳贻贝whirlin类贝壳基质蛋白的重组表达与功能分析

贝壳历来是生物工程和材料学研究的重要对象。贝壳中的贝壳基质蛋白质在贝壳的形成与发育过程中具有重要的调控作用。Whirlin类蛋白质(Whirlin-like protein,WLP)是一种从厚壳贻贝(Mytilus coruscus)中鉴定的新型贝壳基质蛋白质。序列分析结果显示,该蛋白质含有PDZ(postsynaptic density/Discs large/Zonula occludens)结构域,而该结构域对贝壳生物矿化的影响目前尚无报道。为深入了解WLP在贝壳形成中对碳酸钙晶体的影响,在序列分析基础上,采用密码子优化结合原核重组表达,获得其重组表达产物后,开展了重组WLP对碳酸钙晶体形貌及晶型的影响研究,结晶速度抑制以及碳酸钙晶体结合分析。分析结果表明,重组WLP能诱导文石型碳酸钙晶体的形貌和方解石型碳酸钙晶体的晶型发生改变;同时重组WLP对碳酸钙晶体具有结合作用,且能抑制碳酸钙晶体的结晶速度。上述结果表明,WLP对贝壳的形成及发育具有重要影响,并可能在贝壳肌棱柱层的形成中发挥了重要作用。     

Intrinsically Disordered and Pliable Starmaker-Like Protein from Medaka (Oryzias latipes) Controls the Formation of Calcium Carbonate Crystals

Fish otoliths, biominerals composed of calcium carbonate with a small amount of organic matrix, are involved in the functioning of the inner ear. Starmaker (Stm) from zebrafish (Danio rerio) was the first protein found to be capable of controlling the formation of otoliths. Recently, a gene was identified encoding the Starmaker-like (Stm-l) protein from medaka (Oryzias latipes), a putative homologue of Stm and human dentine sialophosphoprotein. Although there is no sequence similarity between Stm-l and Stm, Stm-l was suggested to be involved in the biomineralization of otoliths, as had been observed for Stm even before. The molecular properties and functioning of Stm-l as a putative regulatory protein in otolith formation have not been characterized yet. A comprehensive biochemical and biophysical analysis of recombinant Stm-l, along with in silico examinations, indicated that Stm-l exhibits properties of a coil-like intrinsically disordered protein. Stm-l possesses an elongated and pliable structure that is able to adopt a more ordered and rigid conformation under the influence of different factors. An in vitro assay of the biomineralization activity of Stm-l indicated that Stm-l affected the size, shape and number of calcium carbonate crystals. The functional significance of intrinsically disordered properties of Stm-l and the possible role of this protein in controlling the formation of calcium carbonate crystals is discussed.     

Potential soil reinforcement by biological denitrification

Currently new ground reinforcement techniques are being developed based on microbially induced carbonate precipitation (MICP). Many studies on MICP use microbially catalyzed hydrolysis of urea to produce carbonate. In the presence of dissolved calcium this process leads to precipitation of calcium carbonate crystals, which form bridges between the sand grains and hence increase strength and stiffness. In addition to urea hydrolysis, there are many other microbial processes which can lead to the precipitation of calcium carbonate. In this study the theoretical feasibility of these alternative MICP processes for ground reinforcement is evaluated. Evaluation factors are substrate solubility, CaCO₃ yield, reaction rate and type and amount of side-product. The most suitable candidate as alternative MICP method for sand consolidation turned out to be microbial denitrification of calcium nitrate, using calcium salts of fatty acids as electron donor and carbon source. This process leads to calcium carbonate precipitation, bacterial growth and production of nitrogen gas and some excess carbon dioxide. The feasibility of MICP by denitrification is tested experimentally in liquid batch culture, on agar plate and in sand column experiments. Results of these experiments are presented and discussed.