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

Abstract

In light of increasing sea surface temperatures, quantifying the expression of stressinducible 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 α-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°C) for 1, 3, 14 and 15 d. A rapid down-regulation of most genes (actinrelated 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°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°C only survived for the first 3 d, none of the genes in sponges kept at 31°C were significantly different from those in the 27°C controls after 14 d. This indicates a very strict thermal threshold for R. odorabile between 31 and 32°C and is consistent with previous findings based on sponge necrosis and symbiotic disruptions in this species.KEY WORDS: qPCR · Quantitative PCR · Thermal stress · Porifera · Great Barrier Reef Resale or republication not permitted without written consent of the publisherMar Ecol Prog Ser 431: [97][98][99][100][101][102][103][104][105] 2011 tance of Porifera, the effect of thermal stress on sponges has rarely been investigated, particularly at the molecular level. The few studies published on this topic include a thermal-stress experiment in which Western blotting was utilised to show that the marine sponge Suberites domuncula ex presses a 45 kDa polypeptide after heat treatment (Bachinski et al. 1997). Moreover, a drop in trehalose (a disaccharide that can protect yeast from temperature stress as shown by Hottiger et al. 1987 andEleutherio et al. 1993), a reduction in the activity of the detoxifying and antioxidant enzyme glutathione-S-transferase (GST) and a concomitant decrease of its substrate glutathione (GSH) were recorded in the same specimens. In heatand cold-stressed Geodia cydonium, an in creased transcription of the heat shock protein 70 (Hsp70) gene and a reduction of the Rab GDP dissociation inhibitor (GDI) mRNA (a key element in the intracellular traffic system) were observed using Northern blotting (Krasko et al. 1997). These findings indicate the presence of active heat-stress protection mechanisms in sponges at the cellular leve...

Marine microbial symbiosis heats up: the phylogenetic and functional response of a sponge holobiont to thermal stress

“…Expression of the Hsp 70 and apoptosis-linked genes increased in sponges at 32 1C when compared with healthy sponges, demonstrating a direct molecular response to heat stress (Supplementary Table S1). These results are consistent with our recent studies of thermal stress responses in R. odorabile during different life stages based on quantitative PCR (Pantile and Webster, 2011) and multiplexed reverse transcription quantitative PCR (Webster et al, In Review). In these two studies, the immediate cellular response to thermal stress was also an increased production of Hsp transcripts, which, due to the energetic cost of expression, might compromise normal cellular functions (Feder and Hofmann, 1999).…”

“…The set of 23 genes were selected following screening of a previously prepared R. odorabile complementary DNA clone library (Pantile and Webster, 2011) on the basis of their known or putative role in the cell stress response and cellular homeostasis-related processes (Supplementary Table S1). The kanA gene conferring kanamycin resistance was used as the internal control.…”

Stress levels over time in the introduced ascidian Styela plicata: the effects of temperature and salinity variations on hsp70 gene expression

“…High levels of hsp70 gene expression have been correlated with seawater temperature increases in many marine invertebrates (Osovitz and Hofmann 2005;López-Legentil et al 2008;Pantile and Webster 2011). Accordingly, in this study, we found that significantly higher levels of hsp70 gene expression occurred during the summer months.…”

Sponge-Microbe Associations Survive High Nutrients and Temperatures

“…Previous studies assessing the response of R. odorabile to thermal stress have indicated sub-lethal effects at 31°C, including activation of the heat shock protein system [68] and a significant decrease in flow rate, filtration efficiency and choanocyte chamber density [115]. Whilst the bacterial community of R. odorabile is highly stable at 31°C, higher seawater temperatures cause a shift in the symbiont community which is concomitant with host tissue necrosis and mortality after four days at 32°C or three days at 33°C [67], [69].…”

“…Elevated seawater temperature has previously been shown to cause a shift in the dominant microbial community on marine sponges as well as a decline in sponge health [62]–[64], with some mass mortality events concomitant with anomalies in sea surface temperature [65], [66]. Previous experiments have demonstrated that adult R. odorabile exhibit necrosis and a loss of microbial symbionts within 72 h at 33°C [67], with subsequent experiments confirming a narrow thermal threshold for the host and symbiont community between 31–32°C [68], [69]. Here we investigated the combined effects of water quality and elevated seawater temperature by exposing sponges to a range of elevated nutrient levels under ambient (27°C) and sub-lethal (31°C) seawater temperatures.…”