Elucidating the Functions of Proteins Up-regulated During Diapause in Artemia franciscana Using RNAi

Abstract

Diapause embryos of the animal extremophile Artemia franciscana, a crustacean, are metabolically dormant and exceptionally tolerant to stressors such as heat and anoxia, characteristics thought to depend on the protective activity of molecular chaperones. RNAi methodology was developed and used to knock down individual molecular chaperones which are normally up-regulated during Artemia diapause. DsRNA and siRNAs injected into females were effective in knocking down proteins in embryos into the fifth release. Five proteins were knocked down including the ?-crystallin related small heat shock proteins (sHsps) p26, ArHsp21 and ArHsp22, artemin, a species-specific chaperone as well as p8 a transcription co-factor. The individual sHsps, artemin and p8 exhibited different roles during cyst development and diapause with only some of these proteins contributing to stress tolerance. p26, for example, enhances stress resistance in Artemia embryos, facilitates embryo development and prevents diapause termination indicated by spontaneous hatching. ArHsp21, another ?-crystallin type small heat shock protein contributes only slightly to freezing and desiccation stress and is not protective during heat stress. DsRNA specific to ArHsp22 is lethal to both male and female adults. Artemin contributes to stress tolerance but to a lesser extent than p26. Artemin also extended the period of time over which cysts were released. Cysts that did not contain p8 were also less stress resistant than those that did contain p8 and hatched upon release 10% for the time, suggesting an important role in diapause. By revealing separate and novel roles for molecular chaperones this work contributes substantially to our understanding of diapause, an important, phylogenetically widespread developmental process.