New insights into the anhydrobiotic role of trehalose biosynthesis and transport in <i>Artemia franciscana</i> diapause embryos

Trehalose accumulation is commonly observed in extremophile organisms capable of surviving extended periods of complete desiccation and freezing. However, direct evidence linking the sugar to cryptobiotic survival in metazoans is generally lacking. Here, using the extremophile crustacean Artemia franciscana as a model, we test the anhydrobiotic and cryobiotic roles of trehalose through RNAi-mediated knockdown of its biosynthetic and transport pathways. Transcriptomic and genomic screening uncovered a major expansion of trehalose transporter (tret) genes in branchiopods with up to 27 copies in Artemia sp., but only a single trehalase enzyme (treh), and three trehalose-6-phosphate synthase (tps) genes containing fused synthase and phosphatase domains. Gene expression analysis determined that the tps and selected tret genes are expressed within the diapause-programmed oocytes and embryos, with independent RNAi-mediated knockdown of each reducing cyst trehalose accumulation by approximately 90% and 30%, respectively. Trehalose depletion in diapause cysts critically affects the anhydrobiotic and cryobiotic survival of embryos, a feature confirmed through diapause-termination experiments using either desiccation or H2O2 exposure. These latter data further uncovered the developmental reprogramming of carbohydrate metabolism to cope with low amounts of stored trehalose in the cysts. The findings demonstrate that diapause-induced biosynthesis and transport of trehalose are essential for A. franciscana anhydrobiosis.