Chitin-binding Proteins Of Artemia Participate In Formation Of The Diapause Cysts

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. Large numbers of diapause-related proteins are differentially expressed in embryos from oviparous and ovoviviparous Artemia.The genomic comparison of oviparous and ovoviviparous ovisacs was described previously. We isolated three genes that are significantly up-regulated in oviparous oocytes by screening a subtracted cDNA library created from oviparous and ovoviviparous ovisacs, and identified them as Arp-CBP genes(Artemia parthenogenetica chitin-binding protein gene). Quantitative RT-PCR indicated that the expression of Arp-CBP genes gradually increases during diapause cyst formation in oviparous Artemia and that significant accumulation of Arp-CBP mRNA 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 embryonic cuticle layer of the cyst shell. Furthermore, knock-down of Arp-CBP by RNAi 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 cuticular membrane and outer cuticular membrane 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 embryonic cuticle layer 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 embryonic cuticle layer of the cyst shell.