title: 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.
authors.affiliation: Institute of Cell Biology and Genetics, College of Life Sciences, Zijingang Campus, Zhejiang University, Hangzhou, Zhejiang, People's Republic of China.
authors.forename: Wen-Ming
authors.initials: WM
authors.lastname: Ma
authors.forename: Hua-Wei
authors.initials: HW
authors.lastname: Li
authors.forename: Zhong-Min
authors.initials: ZM
authors.lastname: Dai
authors.forename: Jin-Shu
authors.initials: JS
authors.lastname: Yang
authors.forename: Fan
authors.initials: F
authors.lastname: Yang
authors.forename: Wei-Jun
authors.initials: WJ
authors.lastname: Yang
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D001156:Artemia / Q000196:embryology* / Q000235:genetics / Q000378:metabolism*; D002352:Carrier Proteins / Q000235:genetics / Q000378:metabolism*; D002686:Chitin / Q000235:genetics / Q000378:metabolism*; D004625:Embryo, Nonmammalian / Q000196:embryology* / Q000378:metabolism*; D008969:Molecular Sequence Data
chemical_list: D002352:Carrier Proteins; D002686:Chitin