Chitin-binding proteins of Artemia diapause cysts participate in formation of the embryonic cuticle layer of cyst shells

Abstract

The brine shrimp Artemia reproduces either ovoviviparously, producing free-swimming nauplii, or oviparously, producing encysted embryos (diapause cysts) able to cope with harsh and complex habitats. When the cysts enter diapause they are encased in a complex external shell that protects them from certain extreme environments. The genomic comparison of oviparous and ovoviviparous ovisacs has been described previously. We isolated three significantly up-regulated genes in oviparous oocytes and identified them as Arp-CBP (Artemia parthenogenetica chitin-binding protein) genes. Quantitative real-time PCR indicated that the expression of Arp-CBP genes gradually increases during diapause cyst formation and significant mRNA accumulation occurs during the ovisac stage of oviparous development. Moreover, in situ hybridization results demonstrated that Arp-CBP mRNAs are expressed in the embryo. Interestingly, the results of immune electron microscopy showed that all three Arp-CBPs are distributed throughout the cellular ECL (embryonic cuticle layer) of the cyst shell. Furthermore, knockdown of Arp-CBP by RNA interference resulted in marked changes in the composition of the embryonic cuticular layer. The fibrous layer of the cyst shell adopted a loose conformation and the inner and outer cuticular membranes exhibited marked irregularities when Arp-CBP expression was suppressed. Finally, an in vitro recombinant protein-binding assay showed that all three Arp-CBPs have carbohydrate-binding activities. These findings provide significant insight into the mechanisms by which the ECL of Artemia cyst shell is formed, and demonstrate that Arp-CBPs are involved in construction of the fibrous lattice and are required for formation of the ECL of the cyst shell.

Artemin, a Diapause-Specific Chaperone, Contributes to the Stress Tolerance of Artemia Cysts and Influences Their Release from Females

“…Ovoviviparously developing embryos of Artemia franciscana Kellogg 1906 emerge from females as swimming nauplii, whereas oviparous development yields gastrulae encased in chitinous shells, termed cysts (Jackson and Clegg, 1996;Liang and MacRae, 1999;MacRae, 2003;Ma et al, 2013) (Fig. 1).…”

Three dimensional (3D) structure prediction and substrate-protein interaction study of the chitin binding protein CBP24 from B. thuringiensis

“…Three chitin binding proteins (CBPs) from Artemia parthenogenetica (a shrimp) have been found involved in oviparous development. Moreover, it was found that these proteins are involved in the embryonic cuticular layer and all three CBPs have shown carbohydrate-binding activities [15]. Nonetheless, chitin binding proteins have been found involved in adhesion strategy of some pathogenic bacteria, including E. coli, Listeria monocytogenes and Vibrio cholerae during the intestinal inflammation in humans [16].…”

Domain wise docking analyses of the modular chitin binding protein CBP50 from Bacillus thuringiensis serovar konkukian S4

“…On the other hand, some chitin binding proteins have shown synergistic action with chitinases either specifically or non-specifically [4]. They are believed to have important roles such as antifungal activity, oviparous and cuticular layer development, biosynthesis of fungal cell walls and anti-inflammatory actions [5, 6]. Moreover, they have been found involved in adhesion strategy of some pathogenic bacteria [7].…”