The Roles of Heat Shock Factor during Diapause and Stress Tolerance in the Crustacean Artemia franciscana

Abstract

Encysted embryos of the crustacean Artemia franciscana enter diapause, a developmental state of greatly reduced metabolic activity and enhanced stress tolerance that allows survival under adverse environmental conditions. Diapausing A. franciscana are characterized by the synthesis of molecular chaperones such as the small heat shock proteins (sHSPs), p26, ArHsp21 and ArHsp22, and the ferritin homologue, artemin. A full-length cDNA encoding the A. franciscana transcription factor, heat shock factor 1 (Hsf1), was cloned and sequenced, revealing a deduced protein of 65.3 kDa which contained a conserved DNA binding domain. Hsf1 cDNA was employed as template to make double stranded RNA (dsRNA) used for RNA interference (RNAi) and thus to determine the role of Hsf1 in the regulation of gene expression during diapause. qRT-PCR and immunoprobing of western blots with an antibody raised to Hsf1 from A. franciscana respectively demonstrated the knockdown of hsf1 mRNA and Hsf1 in nauplii and cysts released from females receiving dsRNA. Nauplii lacking Hsf1 appeared normal but they died prematurely upon culturing. Hsf1 knockdown cysts were less stress resistant than cysts containing Hsf1, demonstrating a role for this transcription factor in the stress tolerance of diapausing A. franciscana. Immunoprobing of western blots showed that cysts released from females receiving hsf1 dsRNA lacked p26, an important diapause-specific sHsp in A. franciscana cysts. The knockdown of other sHsps and artemin, which have less influence on the stress tolerance of diapause cysts, also occurred but was more variable in extent. qRT-PCR and immunoprobing of western blots with an antibody raised to Hsf1 from A. franciscana demonstrates hsf1 mRNA and Hsf1 appeared earlier and was more abundant in oviparous versus ovoviviparous embryos. Examination of immunofluoresencently stained samples demonstrates Hsf1 localized earlier to nuclei from oviparous embryos. The results support the conclusion that Hsf1, perhaps in concert with other transcription factors, regulates expression of diapause-specific genes required for stress resistance and diapause in A. franciscana and that Hsf1 is important during A. franciscana development especially for the oviparous embryos.