The Molecular Mechanism Of Formation Of Diapause Cyst Shell In Brine Shrimp, Artemia Parthenogenetica And Its Resistance Role In Environmental Stresses

Artemia, a small crustacean which live in salt lakes on plateaus, has attracted much attention for its special reproduction pattern and surprising stress tolerance. To cope with harsh and complex habitats such as salt lakes, Artemia are able, when the circumstances become adverse, to release their offspring into a dormant, encysted state, rather than simply releasing swimming nauplius in order to ensure survival. The diapause cysts are wrapped in a noncellular rigid shell. They have the capability to withstand the greatest environmental stress, including complete desiccation, long-term anoxia, temperature extremes and ultraviolet-irradiation, then resume development in appropriate conditions. Previous experimental data has confirmed the protective capabilities of the cyst shell. However what the cyst shell is synthesized from and how the formative process is performed remains, as yet, largely unknown.In our experiments, over twenty oviparous specifically expressed genes were identified through screening the subtracted cDNA library enriched between oviparous and ovoviviparous Artemia ovisacs. Among of them, a shell gland-specifically expressed gene (SGEG) has been found to be involved in the cyst shell formation. SGEG contains a 345 bp open-reading frame, and its consequentially translated peptide consists of a 33 amino acid residue putative signal peptide and an 81 amino acid residue mature peptide. It was considered to be a novel gene, having no apparent identity with other known genes or proteins when alignments were compared to sequences in the DDBJ/EMBL/GenBank database.The results of Northern blotting and in situ hybridization indicate that the gene is specifically expressed in the cells of shell glands during the period of diapause cyst formation of oviparous Artemia. Also, the mRNA of SGEG is only abundantly accumulated when the periods of ovulation and the oocytes temporary stored in lateral pouches.Lacking SGEG protein (by RNA interference) caused the cyst shell to become translucent and the chorion layer of the shell become less compact and pultaceous, and to show a marked decrease of iron composition within the shell the kalium content was higher. For the RNAi-induced cysts, more than 20% were found to be able to develop into swimming nauplii without any activation necessary. Moreover, several specific proteins of RNAi-induced cysts were downregulated while several proteins were upregulated. All these aspects imply an incomplete diapause of RNAi-induced cysts, and would lead to a continuation of development a short time after oviposition.More interestingly, the RNA interference induced defective diapause cysts with a totally compromised resistibility to UV irradiation, extremely large temperature differences, osmotic pressure, dryness and organic solvent stresses. In contrast, the control cysts maintained a normal hatching rate after such environmental stresses.In conclusion, this investigation provides evidence increasing our understanding of the study of cyst shell formation of Artemia, and may provide a new insight into other related research in extremophile biology. Thereafter, we may develop a new excellent biomaterial owe to the special cyst shell characteristics, and the affected cyst may act as a convenient weaning food of larvae.