Assaf Shechter

A gastrolith protein serving a dual role in the formation of an amorphous mineral containing extracellular matrix

Despite the proclamation of Lowenstam and Weiner that crustaceans are the “champions of mineral mobilization and deposition of the animal kingdom,” relatively few proteins from the two main calcification sites in these animals, i.e., the exoskeleton and the transient calcium storage organs, have been identified, sequenced, and their roles elucidated. Here, a 65-kDa protein (GAP 65) from the gastrolith of the crayfish, Cherax quadricarinatus, is fully characterized and its function in the mineralization of amorphous calcium carbonate (ACC) of the extracellular matrix is demonstrated. GAP 65 is a negatively charged glycoprotein that possesses three predicted domains: a chitin-binding domain 2, a low-density lipoprotein receptor class A domain, and a polysaccharide deacetylase domain. Expression of GAP 65 was localized to columnar epithelial cells of the gastrolith disk during premolt. In vivo administration of GAP 65 dsRNA resulted in a significant reduction of GAP 65 transcript levels in the gastrolith disk. Such gene silencing also caused dramatic structural and morphological deformities in the chitinous-ACC extracellular matrix structure. ACC deposited in these gastroliths appeared to be sparsely packed with large elongated cavities compared with the normal gastrolith, where ACC is densely compacted. ACC spherules deposited in these gastroliths are significantly larger than normal. GAP 65, moreover, inhibited calcium carbonate crystallization in vitro and stabilized synthetic ACC. Thus, GAP 65 is the first protein shown to have dual function, involved both in extracellular matrix formation and in mineral deposition during biomineralization.

Reciprocal Changes in Calcification of the Gastrolith and Cuticle During the Molt Cycle of the Red Claw Crayfish Cherax quadricarinatus

Mobilization of calcium during the molt cycle from the cuticle to transient calcium deposits is widely spread in crustaceans. The dynamics of calcium transport to transient calcium deposits called gastroliths and to the cuticle over the course of the molt cycle were studied in the crayfish Cherax quadricarinatus. In this species, calcium was deposited in the gastroliths during premolt and transported back to the cuticle during postmolt, shown by digital X-ray radiograph analysis. The predominant mineral in the crayfish is amorphous calcium carbonate embedded in an organic matrix composed mainly of chitin. Scanning electron micrographs of the cuticle during premolt showed that the endocuticle and parts of the exocuticle were the source of most of the labile calcium, while the epicuticle did not undergo degradation and remained mineralized throughout the molt cycle. The gastroliths are made of concentric layers of amorphous calcium carbonate intercalated between chitinous lamella. Measurements of pH and calcium levels during gastrolith deposition showed that calcium concentrations in the gastroliths, stomach, and muscle were about the same (10 to 11 mmol l−1). On the other hand, pH varied greatly, from 8.7 ± 0.15 in the gastrolith cavity through 7.6 ± 0.2 in muscle to 6.9 ± 0.5 in the stomach.

Search for hepatopancreatic ecdysteroid-responsive genes during the crayfish molt cycle: from a single gene to multigenicity

SUMMARY The expression of the vitellogenin gene of the red-claw crayfish Cherax quadricarinatus (CqVg) was previously demonstrated in male crayfish during an endocrinologically induced molt cycle. The hypothesis that this expression is under the direct control of ecdysteroids was tested in this study both in vivo and in vitro. Unlike vitellogenin of insects, CqVg was not found to be ecdysteroid-responsive. Thus, a multigenic approach was employed for the identification of other hepatopancreatic ecdysteroid-responsive genes by a cDNA microarray. For the purposes of this study, a multi-parametric molt-staging technique, based on X-ray detection of gastrolith growth, was developed. To identify ecdysteroid-responsive genes during premolt, the molt cycle was induced by two manipulations, 20-hydroxyecdysone administration and X-organ–sinus gland complex removal; both resulted in significant elevation of ecdysteroids. Two clusters of affected genes (129 and 122 genes, respectively) were revealed by the microarray. It is suggested that only genes belonging to similarly responsive (up- or downregulated) gene clusters in both manipulations (102 genes) could be considered putative ecdysteroid-responsive genes. Some of these ecdysteroid-responsive genes showed homology to genes controlling chitin metabolism, proteases and other cellular activities, while 56.8% were unknown. The majority of the genes were downregulated, presumably by an energetic shift of the hepatopancreas prior to ecdysis. The effect of 20-hydroxyecdysone on representative genes from this group was confirmed in vitro using a hepatopancreas tissue culture. This approach for ecdysteroid-responsive gene identification could also be implemented in other tissues for the elucidation of ecdysteroid-specific signaling pathways during the crustacean molt cycle.

A Protein Involved in the Assembly of an Extracellular Calcium Storage Matrix

Gastroliths, the calcium storage organs of crustaceans, consist of chitin-protein-mineral complexes in which the mineral component is stabilized amorphous calcium carbonate. To date, only three proteins, GAP 65, gastrolith matrix protein (GAMP), and orchestin, have been identified in gastroliths. Here, we report a novel protein, GAP 10, isolated from the gastrolith of the crayfish Cherax quadricarinatus and specifically expressed in its gastrolith disc. The encoding gene was cloned by partial sequencing of the protein extracted from the gastrolith matrix. Based on an assembled microarray cDNA chip, GAP 10 transcripts were found to be highly (12-fold) up-regulated in premolt gastrolith disc and significantly down-regulated in the hypodermis at the same molt stage. The deduced protein sequence of GAP 10 lacks chitin-binding domains and does not show homology to known proteins in the GenBankTM data base. It does, however, have an amino acid composition that has similarity to proteins extracted from invertebrate and ascidian-calcified extracellular matrices. The GAP 10 sequence contains a predicted signal peptide and predicted phosphorylation sites. In addition, the protein is phosphorylated and exhibits calcium-binding ability. Repeated daily injections of GAP 10 double strand RNA to premolt C. quadricarinatus resulted in a prolonged premolt stage and in the development of gastroliths with irregularly rough surfaces. These findings suggest that GAP 10 may be involved in the assembly of the gastrolith chitin-protein-mineral complex, particularly in the deposition of amorphous calcium carbonate.

Hepatopancreatic multi-transcript expression patterns in the crayfish Cherax quadricarinatus during the moult cycle

Alterations of hepatopancreatic multi‐transcript expression patterns, related to induced moult cycle, were identified in male Cherax quadricarinatus through cDNA microarray hybridizations of hepatopancreatic transcript populations. Moult was induced by X–organ sinus gland extirpation or by repeated injections of 20‐hydroxyecdysone. Manipulated males were sacrificed at premoult or early postmoult, and a reference population was sacrificed at intermoult. Differentially expressed genes among the four combinations of two induction methods and two moult stages were identified. Biologically interesting clusters revealing concurrently changing transcript expressions across treatments were selected, characterized by a general shift of expression throughout premoult and early postmoult vs. intermoult, or by different premoult vs. postmoult expressions. A number of genes were differentially expressed in 20‐hydroxyecdysone‐injected crayfish vs. X–organ sinus gland extirpated males.

Solubility and Bioavailability of Stabilized Amorphous Calcium Carbonate

Since its role in the prevention of osteoporosis in humans was proven some 30 years ago, calcium bioavailability has been the subject of numerous scientific studies. Recent technology allowing the production of a stable amorphous calcium carbonate (ACC) now enables a bioavailability analysis of this unique form of calcium. This study thus compares the solubility and fractional absorption of ACC, ACC with chitosan (ACC‐C), and crystalline calcium carbonate (CCC). Solubility was evaluated by dissolving these preparations in dilute phosphoric acid. The results demonstrated that both ACC and ACC‐C are more soluble than CCC. Fractional absorption was evaluated by intrinsically labeling calcium carbonate preparations with 45Ca, orally administrated to rats using gelatin capsules. Fractional absorption was determined by evaluating the percentage of the administrated radioactive dose per milliliter that was measured in the serum, calcium absorption in the femur, and whole‐body retention over a 34‐hour period. Calcium serum analysis revealed that calcium absorption from ACC and ACC‐C preparations was up to 40% higher than from CCC, whereas retention of ACC and ACC‐C was up to 26.5% higher than CCC. Absorbed calcium in the femurs of ACC‐administrated rats was 30% higher than in CCC‐treated animals, whereas 15% more calcium was absorbed following ACC‐C treatment than following CCC treatment. This study demonstrates the enhanced solubility and bioavailability of ACC over CCC. The use of stable ACC as a highly bioavailable dietary source for calcium is proposed based on the findings of this study.

Multi-transcript expression patterns in the gastrolith disk and the hypodermis of the crayfish Cherax quadricarinatus at premolt

In the crustacean Cherax quadricarinatus, alterations of multi-transcript expression patterns between intermolt and late premolt stages were identified in the hypodermis and in the gastrolith disk via a cDNA microarray. The gastrolith disk is a specialized epithelium forming the gastroliths at premolt. The gastroliths are deposits of calcium carbonate derived from the digested cuticle contributing the mineral to the newly formed exoskeleton at postmolt. The late premolt stage was characterized by a dramatic general up-regulation of genes in the gastrolith disk. This phenomenon is explained by the gastrolith disk function rapid formation of the relatively large gastrolith during a short period of time. Besides genes of general importance for this dramatic change, three genes related to the chitin-protein-mineral structure were identified. The cDNA and the deduced protein of the novel one of them, the chitin deacetylase 1 (Cq-CDA1) was fully characterized and its resemblance to already characterized structural proteins of the gastrolith matrix was described. Cq-CDA1 characteristics strongly indicate its participation in the gastrolith construction, although its protein product was not identified yet in the gastrolith. In addition, many differentially expressed genes with unknown function were elucidated. An unexpected milder down-regulation was observed in the hypodermis.

A Single Injection of Hypertrophied Androgenic Gland Cells Produces All-Female Aquaculture

Monosex culture, common in animal husbandry, enables gender-specific management. Here, production of all-female prawns (Macrobrachium rosenbergii) was achieved by a novel biotechnology comprising three steps: (a) A single injection of suspended hypertrophied androgenic gland cells caused fully functional sex reversal of females into “neo-males” bearing the WZ genotype; (b) crossing neo-males with normal females (WZ) yielded genomically validated WW females; and (c) WW females crossed with normal males (ZZ) yielded all-female progeny. This is the first sustainable biotechnology for large-scale all-female crustacean aquaculture. The approach is particularly suited to species in which females are superior to males and offers seedstock protection, thereby ensuring a quality seed supply. Our technology will thus revolutionize not only the structure of the crustacean aquaculture industry but can also be applied to other sectors. Finally, the production of viable and reproducible females lacking the Z chromosome questions its role, with respect to sexuality.

Increased calcium absorption from synthetic stable amorphous calcium carbonate: double-blind randomized crossover clinical trial in postmenopausal women.

Calcium supplementation is a widely recognized strategy for achieving adequate calcium intake. We designed this blinded, randomized, crossover interventional trial to compare the bioavailability of a new stable synthetic amorphous calcium carbonate (ACC) with that of crystalline calcium carbonate (CCC) using the dual stable isotope technique. The study was conducted in the Unit of Clinical Nutrition, Tel Aviv Sourasky Medical Center, Israel. The study population included 15 early postmenopausal women aged 54.9 ± 2.8 (mean ± SD) years with no history of major medical illness or metabolic bone disorder, excess calcium intake, or vitamin D deficiency. Standardized breakfast was followed by randomly provided CCC or ACC capsules containing 192 mg elemental calcium labeled with 44Ca at intervals of at least 3 weeks. After swallowing the capsules, intravenous CaCl2 labeled with 42Ca on was administered on each occasion. Fractional calcium absorption (FCA) of ACC and CCC was calculated from the 24‐hour urine collection following calcium administration. The results indicated that FCA of ACC was doubled (± 0.96 SD) on average compared to that of CCC (p < 0.02). The higher absorption of the synthetic stable ACC may serve as a more efficacious way of calcium supplementation.