Strict thermal threshold identified by quantitative PCR in the sponge Rhopaloeides odorabile

In light of increasing sea surface temperatures, quantifying the expression of stress-inducible genes in coastal organisms is imperative to identify early biomarkers of thermal stress. In the present study we developed a quantitative PCR (qPCR) assay to test the molecular response to heat stress in the Great Barrier Reef sponge Rhopaloeides odorabile. Suitable reference genes (coding for a-tubulin, 28S rRNA and ubiquitin) were identified among 5 candidates and then used to normalise expression of target genes (actin-related protein, calmodulin, ferritin, ubiquitin-conjugating enzyme, heat shock protein 90 [Hsp90] and heat shock protein 40 [Hsp40]) in samples exposed to high temperatures (31 and 32 degrees C) for 1, 3, 14 and 15 d. A rapid down-regulation of most genes (actin-related protein, ferritin, calmodulin and Hsp90) was observed at both temperatures within 24 h, indicating an initial shut-down of the sponge's molecular systems in response to thermal stress. The increased expression of Hsp40 and Hsp90 in sponges at 32 degrees C after 1 and 3 d respectively indicates an activation of the heat shock response system and is consistent with their role as chaperones for directing degraded proteins to proteolysis, this last process being sustained by an induction of the ubiquitin-conjugating enzyme gene at this temperature. While sponges kept at 32 degrees C only survived for the first 3 d, none of the genes in sponges kept at 31 degrees C were significantly different from those in the 27 degrees C controls after 14 d. This indicates a very strict thermal threshold for R. odorabile between 31 and 32 degrees C and is consistent with previous findings based on sponge necrosis and symbiotic disruptions in this species.