Calcium phosphate mineralization is widely applied in crustacean mandibles

© The Author(s), 2016. This article is distributed under the terms of the Creative Commons Attribution License. The definitive version was published in Scientific Reports 6 (2016): 22118, doi:10.1038/srep22118.Crustaceans, like most mineralized invertebrates, adopted calcium carbonate mineralization for bulk skeleton reinforcement. Here, we show that a major part of the crustacean class Malacostraca (which includes lobsters, crayfishes, prawns and shrimps) shifted toward the formation of calcium phosphate as the main mineral at specified locations of the mandibular teeth. In these structures, calcium phosphate is not merely co-precipitated with the bulk calcium carbonate but rather creates specialized structures in which a layer of calcium phosphate, frequently in the form of crystalline fluorapatite, is mounted over a calcareous “jaw”. From a functional perspective, the co-existence of carbonate and phosphate mineralization demonstrates a biomineralization system that provides a versatile route to control the physico-chemical properties of skeletal elements. This system enables the deposition of amorphous calcium carbonate, amorphous calcium phosphate, calcite and apatite at various skeletal locations, as well as combinations of these minerals, to form graded composites materials. This study demonstrates the widespread occurrence of the dual mineralization strategy in the Malacostraca, suggesting that in terms of evolution, this feature of phosphatic teeth did not evolve independently in the different groups but rather represents an early common trait.This study was supported in part by grants from the Israel Science Foundation (ISF, Grant 613/13) and the National Institute for Biotechnology in the Negev (NIBN)

The role of seawater endocytosis in the biomineralization process in calcareous foraminifera

Foraminifera are unicellular organisms that inhabit the oceans in various ecosystems. The majority of the foraminifera precipitate calcitic shells and are among the major CaCO3 producers in the oceans. They comprise an important component of the global carbon cycle and also provide valuable paleoceanographic information based on the relative abundance of stable isotopes and trace elements (proxies) in their shells. Understanding the biomineralization processes in foraminifera is important for predicting their calcification response to ocean acidification and for reliable interpretation of the paleoceanographic proxies. Most models of biomineralization invoke the involvement of membrane ion transporters (channels and pumps) in the delivery of Ca2+ and other ions to the calcification site. Here we show, in contrast, that in the benthic foraminiferan Amphistegina lobifera, (a shallow water species), transport of seawater via fluid phase endocytosis may account for most of the ions supplied to the calcification site. During their intracellular passage the seawater vacuoles undergo alkalization that elevates the CO32− concentration and further enhances their calcifying potential. This mechanism of biomineralization may explain why many calcareous foraminifera can be good recorders of paleoceanographic conditions. It may also explain the sensitivity to ocean acidification that was observed in several planktonic and benthic species

A Novel Chitin Binding Crayfish Molar Tooth Protein with Elasticity Properties

<div><p>The molar tooth of the crayfish <i>Cherax quadricarinatus</i> is part of the mandible, and is covered by a layer of apatite (calcium phosphate). This tooth sheds and is regenerated during each molting cycle together with the rest of the exoskeleton. We discovered that molar calcification occurs at the pre-molt stage, unlike calcification of the rest of the new exoskeleton. We further identified a novel molar protein from <i>C</i>. <i>quadricarinatus</i> and cloned its transcript from the molar-forming epithelium. We termed this protein Cq-M13. The temporal level of transcription of <i>Cq-M13</i> in an NGS library of molar-forming epithelium at different molt stages coincides with the assembly and mineralization pattern of the molar tooth. The predicted protein was found to be related to the pro-resilin family of cuticular proteins. Functionally, <i>in vivo</i> silencing of the transcript caused molt cycle delay and a recombinant version of the protein was found to bind chitin and exhibited elastic properties.</p></div

Development of a new molar tooth during an induced molt cycle in an ecdysone-injected male <i>C</i>. <i>quadricarinatus</i> and spatial and temporal expression patterns of the <i>Cq-M13</i> transcript <i>in vitro</i> and <i>in silico</i>.

<p><b>A</b>. Changes in molt mineralization index (MMI) during the induced molt cycle following repetitive injections of ecdysone. The x axis is normalized to days from ecdysis. A dashed line on an isolated mandible represents a cut through which a transverse plane is visible (A-top). <b>B</b>. Visualization of the new molar tooth assembly process in the crayfish mandible on days -9, -8, -7, -4, -3 and 0, following ecdysone injection by <i>ex vivo</i> micro-computed tomography. The top and bottom series of images show a view of the transverse and posterior planes of the mandibles correspondingly. White arrows point to the newly formed molar tooth. <b>C</b>. <i>In silico</i> transcriptomic analysis of <i>Cq-M13</i> read counts from four different molt stages. Different letters above columns represent statistically significant differences (p < 0.05 ± SE). <b>D</b>. Agarose gels showing RT-PCR products demonstrating spatial and temporal expression patterns of the <i>Cq-M13</i> transcript in cuticle-, molar-, basal segment (B.S.)-, maxillae- and gastrolith-forming tissues, as well as in hepatopancreas (Hepato) and testis.</p

Chitin-binding ability of rCq-M13.

<p>Following incubation with chitin powder, equal amounts of Negative control—BSA (top), Positive control—GAP 65 (middle, using gastrolith protein extract with known chitin binding abilities [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0127871#pone.0127871.ref002" target="_blank">2</a>], and rCq-M13 (bottom) underwent three consecutive washes prior to SDS-PAGE. Lane 1 represent the first wash with DDW, lane 2 represent the second wash with NaCl (0.2 M) and lane 3 represent the third wash with denaturation buffer (DB, containing SDS and 2-mercaptoethanol).</p

<i>Cq-M13</i> silencing effects.

<p><b>A</b>. Levels of <i>Cq-M13</i> transcripts following <i>in vivo</i> dsRNA injections in male crayfish, as assessed by real-time RT-PCR following short-term silencing. Animals were injected with ecdysone and either ds<i>Cq-M13</i> or ds<i>CqVg</i>. Different letters represent significant differences and error bars represent standard error (p < 0.05 ± SE). <b>B</b>. Molt cycle progress elongation of <i>Cq-M13</i>-silenced <i>C</i>. <i>quadricarinatus</i> males. The experimental group was injected with <i>Cq-M13</i> dsRNA and the control group was injected with <i>CqVg</i> dsRNA. Both groups were injected with ecdysone to induce their molt cycle. Asterisk represents a significant difference (p < 0.05± SE).</p