Marine Environmental Research 99 (2014) 69e75
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Marine Environmental Research journal homepage: www.elsevier.com/locate/marenvrev
The susceptibility of corals to thermal stress by analyzing Hsp60 expression Davide Seveso a, b, *, Simone Montano a, b, Giovanni Strona c, Ivan Orlandi a, Paolo Galli a, b, Marina Vai a a b c
Department of Biotechnologies and Biosciences, University of Milan e Bicocca, Piazza della Scienza 2, 20126, Milan, Italy MaRHE Centre (Marine Research and High Education Centre), Magoodhoo Island, Faafu Atoll, Maldives European Commission, Joint Research Centre, Institute for Environment and Sustainability, Via E. Fermi 2749, I-21027 Ispra, Italy
a r t i c l e i n f o
a b s t r a c t
Article history: Received 21 March 2014 Received in revised form 23 May 2014 Accepted 13 June 2014 Available online 20 June 2014
Due to the increasing frequency and severity of the coral bleaching events in the context of global warming, there is an urgent need to improve our understanding of the susceptibility of corals to thermal stresses, particularly at the sub-cellular level. In this context, we examined the modulation of the polyp mitochondrial Hsp60 in three scleractinian coral species (Seriatopora hystrix, Montipora monasteriata and Acropora echinata) under simulated heat shock bleaching at 34 C during a time course of 36 h. All three species displayed a similar initial increase of Hsp60 level which accompanies the increasing paleness of coral tissue. Afterwards, each of them showed a specific pattern of Hsp60 down-regulation which can be indicative of a different threshold of resistance, although it proceeded in synchrony with the complete bleaching of tissues. The finely branched S. hystrix was the species most susceptible to heat stress while the plating M. monasteriata was the most tolerant one, as its Hsp60 down-regulation was less rapid than the branching corals. On the whole, the Hsp60 modulation appears useful for providing information about the susceptibility of the different coral taxa to environmental disturbances. © 2014 Elsevier Ltd. All rights reserved.
Keywords: Thermal susceptibility Coral bleaching Heat shock proteins Hsp60 Global climate change
1. Introduction Coral reefs worldwide are among the ecosystems most vulnerable to global climate change (Hughes et al., 2003; Veron et al., 2009). The primary response to hyperthermic stress is represented by coral bleaching events, caused by the breakdown in the relationship between scleractinian corals and their endosymbiotic dinoflagellates (genus Symbiodinium), which in recent decades have led to widespread coral mortality and reef degradation (Weis, 2008; Wilkinson, 2008). Projections about the sea temperature will soon overcome the well-known thermal thresholds for corals, and consequently bleaching events will increasingly become chronic, and affect the long-term viability of coral reefs (Donner et al., 2005; Hoegh-Guldberg et al., 2007). Given the threats of global climate change for coral reefs, there is an urgent need to develop proactive means and biomarkers capable of diagnosing the health of corals
* Corresponding author. Department of Biotechnologies and Biosciences, University of Milan e Bicocca, Piazza della Scienza 2, 20126, Milan, Italy. Tel.: þ39 0264483433. E-mail address: [email protected] (D. Seveso). http://dx.doi.org/10.1016/j.marenvres.2014.06.008 0141-1136/© 2014 Elsevier Ltd. All rights reserved.
before visible and irreparable bleaching takes place (Downs et al., 2000; Mayfield et al., 2011, 2013). The coral heat stress response involves a wide array of cellular and physiological processes, including heterotrophic plasticity, the production of protective pigments, mycosporine-like amino acids and the expression of fluorescent proteins and antioxidant enzymes (Richier et al., 2005; Grottoli et al., 2006; Weis, 2008; Baird et al., 2009). There is also evidence that corals have an enhanced thermotolerance capacity linked to cellular protective mechanisms such as the induction of the Heat shock proteins (Hsps), (Brown et al., 2002; Rosic et al., 2011; Chow et al., 2012). Hsps are ubiquitous molecular chaperones which regulate protein structure and function under physiological conditions, as well as during and after stresses (Hartl et al., 2011). In response to environmental stresses their expression is up-regulated in order to increase cellular repair and tolerance to adverse conditions by preserving metabolic functions (Richter et al., 2010). Among the Hsps, the mitochondrial 60-kDa chaperonin (Hsp60) is an important component of the folding system in the mitochondrial matrix and plays an essential role in both cellular physiology and stress response (Mayer, 2010). The increase in its level indicates a shift in the protein chaperoning and degradation within the mitochondria and implies a change in
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the mitochondrial-associated metabolic pathways (Papp et al., 2003). For these reasons, the expression of the Hsps, such as the Hsp60, can represent potentially useful molecular biomarkers for detecting early signs of change in an organism's physiological state, to the extent that in scleractinian corals the role of Hsp60 as a potential environment stress marker has been widely discussed (Brown et al., 2002; Downs et al., 2005; Rossi et al., 2006; Chow et al., 2009; Seveso et al., 2012, 2013). However, coral susceptibility to stress and bleaching appears to be highly variable since different corals display different levels of physiological resistance to environmental stress (Obura, 2001; McClanahan et al., 2007; Montano et al., 2010). The diversity among species in their susceptibility to disturbances represents a critical aspect of community dynamics, since it can give rise to changes in the community structure and species composition which determine the long-term persistence of coral reefs (Hughes and Connell, 1999). Attempts to understand the differences in the response of reef-building corals to thermal stresses have principally focused on coral morphology, growth and metabolic rates, tissue thickness and host CO2 supply strategies, with faster growing branching taxa being more severely affected by bleaching than slower growing massive taxa (Gates and Edmunds, 1999; Marshall and Baird, 2000; Loya et al., 2001; Darling et al., 2012; Wooldridge, 2014). Although there is an urgent need to understand the factors which determine this susceptibility, until now molecular mechanisms involved in coral thermal tolerance have not been completely elucidated (Weis, 2010), neither has the choice been made of a suitable and fast responding marker for the physiological stress response. In this regard, the use of Hsps as biomarkers to analyze the different susceptibility of reef-building corals to stresses has been partially investigated (Chow et al., 2009; Fitt et al., 2009). In this study we have examined and compared the Hsp60 expression profiles in three different species under simulated heat stress bleaching and during a time course of 36 h. In particular, these analyses were performed in 3 common reef-building scleractinian species belonging to Pocilloporidae (Seriatopora hystrix) and Acroporidae (Montipora monasteriata and Acropora echinata) families, which represent the most sensitive taxa to environmental stresses, since these corals typically showed similar thermal responses and suffered high mortality during bleaching events (Obura, 2005; Montano et al., 2010; Guest et al., 2012). 2. Materials and methods 2.1. Maintenance condition of the corals The coral colonies of S. hystrix, M. monasteriata and A. echinata (n ¼ 3 colonies per species) were maintained under laboratory culture conditions in a flow-through aquaria system at the Civic Aquarium of Milano (Italy) since 2011. The system is composed of 120-l tanks, connected to a 330-l sump containing gravel-bed filter, protein skimmer and a 500 W Titanium Heater (Aqua Medic) with a temperature controller. In all the tanks, the light was supplied by 400 W metal halide lamps (Powerstar HQI-T, Osram), which provided a constant photosynthetically active radiation (PAR) irradiance of 200 ± 30 mmol photons m2 s1 (mE), as reported by (Chow et al., 2009), measured by a photo-radiometer (Delta Ohm e HD2302.0). The photoperiod was set at 12 h:12 h light:dark cycle. Normal water temperature was set at 27 C (±0.2 C) and the 35 ppt of salinity was reproduced. Evaporation in aquaria was offset by the addition of deionized water. Calcium, magnesium, iodium and alkalinity were assessed and reintegrated every two days to ensure stable water chemistry. Salinity and pH of the seawater in aquarium tanks were constantly monitored using a Hach Lange HQ 30D flexi portable meter.
2.2. Experimental design and thermal stress experiment Four nubbins of similar large size were generated with pliers from each of the nine colonies for a total of thirty-six. They were randomly distributed into three aquaria chosen as experimental tanks in order to obtain four nubbins per species per aquarium which represented the biological replicates. For each species nine nubbins (3 per tank) were reserved for Western analyses and three nubbins (1 per tank) for morphological analyses. The coral fragments were maintained in the conditions described above for 10 days and left to recover from the handling procedure until tissue was found to have completely overgrown at the site of fracture (Mayfield et al., 2012). During the acclimatization period at 27 C all the chemical and physical parameters of the seawater were kept stable and constantly monitored. To minimize variations due to light bulb diffusion and water movement, the position of nubbins within each tank was changed every four days. Tanks were cleaned on a weekly basis in order to minimize algal growth (RodolfoMetalpa et al., 2005) and nubbins were visually inspected once a day to check for bleaching and mortality. After the acclimation period, the coral fragments of S. hystrix, M. monasteriata and A. echinata were photographed for morphological analyses and the others were sampled for Western analyses just clipping off a small piece of skeleton with bone-cutting pliers. They represented the control samples (Time 0). After sampling, coral fragments were immediately frozen in liquid nitrogen and stored at 80 C until analysis. Only healthy coral fragments exhibiting healing at fragmentation lesions, growth and dark pigmentation were used as control and for subsequent experiments. Afterwards, all the nubbins of S. hystrix, M. monasteriata and A. echinata were subjected to severe and extreme heat shock at 34 C (±0.2 C) with the aim of inducing coral bleaching and a subcellular thermal stress response at the end of the treatment. For six of the nine nubbins per species reserved for Western analyses, the exposures were carried out for 36 h and the sampling were performed 6, 12, 18, 24, 30 and 36 h after 34 C have been reached. All the samples were immediately frozen in liquid nitrogen and stored at 80 C prior to protein extraction. As the same 18 nubbins were sampled six times, the assumption was made that sampling of a portion of the colony representing less than 0.1% of the total colony volume would not cause stress to another section of the same colony (Mayfield et al., 2011). Furthermore, as artificial lighting was used during the experiments, sampling was performed at the same light level with the exception of the sampling performed after 18 h which was realized during the dark period. After 12 and 36 h at 34 C, a photographic documentation of coral colonies was performed in order to monitor the coral health as tissue coloration and morphological aspect. Moreover, the remaining three nubbins per species reserved for Western analyses after 18 h of thermal stress were returned to 27 C. The sampling of these coral fragments were performed after 12 h and at the end of the recovery treatment. Temperature, PAR, pH, salinity and the other chemical parameters were checked routinely over the course of the experiment. During stress exposures each aquarium tank represented a closed system (static flow) containing Koralia immersed pumps (1200 l/h) in order to continuously mix and aerate the seawater. Elevated temperature was achieved using submersible Acqua Medic Titanium Aquarium Heaters (model TH-100, 240 V) connected to a temperature controller (Eliwell, PC 902T). 2.3. Protein extracts and Western analysis In the laboratory, the frozen coral fragments were powdered using mortar and pestle. Extracts containing only polyp proteins were prepared by homogenization in SDS-buffer, boiling and
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centrifugation steps as previously described (Seveso et al., 2013), in order to remove any Symbiodinium contamination. Moreover, before boiling, for some extracts an equal volume of acid-washed glass beads (425e600 mm, Sigma) was added followed by vigorously shaking on a vortex (10 cycles of 1 min) interspersed with cooling on ice. This latter procedure has been used to prepare extracts containing also Symbiodinium proteins (Weis et al., 2002; Seveso et al., 2013). All the extracts were frozen at 20 C until used. Aliquots were used for protein concentration determinations using the Bio-Rad protein assay kit (Bio-Rad Laboratories). Protein samples were separated by SDS-PAGE on 8% polyacrylamide gels (Vai et al., 1986) and the same amount of proteins was loaded on each lane of the gel. Duplicate gels were run in parallel and after electrophoresis one gel was stained with Coomassie Brilliant Blue to visualize the total proteins and the other electroblotted onto nitrocellulose membrane for Western analysis (Seveso et al., 2012). Correct protein transfer was confirmed by Ponceau S Red (SigmaeAldrich) staining of filters. For each blot, the same amount of recombinant human Hsp60 (Enzo Life Sciences) was included as an internal control for signal differences across blots. Immunodecoration with anti-Hsp60 monoclonal antibody (IgG mouse clone LK-2, SPA-807, Enzo Life Sciences), anti-b-Actin monoclonal antibody (clone C4, MAB1501, Millipore) and secondary antibody anti-mouse IgG conjugated with horseradish peroxidase (Thermo Scientific) was performed as previously reported (Seveso et al., 2013). To evaluate the presence/absence of Symbiodinium proteins, filters were probed with anti-Ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) large subunit antibody (AS03037, Agrisera) followed by incubation with secondary antibody anti-rabbit IgG conjugated with
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horseradish peroxidase (Thermo Scientific) as described (Seveso et al., 2013). Rubisco protein standard (AS01017S, Agrisera) was loaded as internal control. Binding was visualized with the Pierce ECL Western Blotting Substrate followed by X-ray films. 2.4. Densitometric and statistical analyses Densitometric analysis was performed on a Bio-Rad GS-800 calibrated imaging densitometer and quantified with the Image J free software (http://rsb.info.nih.gov/ij/) of the NIH Image software package (National Institutes of Health, Bethesda, Md.). For each blot, the scanned intensity of the Hsp60 bands was normalized against the intensity of the b-Actin ones. Although the housekeeping gene encoding b-Actin has been shown to undergo diel fluctuations in its expression in corals (Mayfield et al., 2010), in all our experiments the b-Actin level did not display a significant modulation under the different exposure time and for each species. Data were expressed as the mean ± standard error of the mean (SEM). Data normality was verified using ShapiroeWilk test. Twoway analysis of variance (ANOVA) was then performed for all the normalized Hsp60 intensity values obtained from the different groups of samples, using species and exposure time as factors. Following two-way ANOVA, a Tukey's HSD post hoc test was performed to assess significant differences (p < 0.05) in Hsp60 protein levels in response to the different exposure times for each species. A non-parametric paired t-test (Wilcoxon signed-rank test) was performed to assess if Hsp60 expression at different times was significantly different in each pair of species.
Fig. 1. Effect of heat shock treatment on coral colonies. Representative photographic documentation of the morphological characteristics of S. hystrix (A), M. monasteriata (B) and A. echinata (C) colonies at 27 C (control, Time 0) and after 12 and 36 h of heat shock at 34 C (scale bar: 1 cm).
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3. Results and discussion Climate change has led to an increase of studies regarding the mechanisms of the stress response at sub-cellular level, especially those that may confer enhanced tolerance to changing environmental conditions for sessile marine taxa, such as scleractinian corals, that cannot easily migrate to new environmental optima (Mayfield and Gates, 2007; Somero, 2012; Doney et al., 2012). In particular, here we focused on the heat shock responses among different coral species during a bleaching event by analyzing and comparing their Hsp60 patterns of expression, with the aim of elucidating the role of Hsps in coral resistance and susceptibility to thermal stresses. To do this, colonies of S. hystrix, M. monasteriata and A. echinata were subjected to an extreme temperature of 34 C for 36 h, starting from a control temperature of 27 C. At first, as expected, morphological analyses of the corals indicated that all the colonies belonging to the three species suffered intense bleaching. In fact, already after 12 h of heat shock all the colonies displayed retracted polyps and pale tissues which became completely bleached at the end of the exposure (Fig. 1). Despite the dissimilar coral growth forms, no significant difference in tissue appearance at the various exposure time was observed among species, which displayed a similar bleaching trend over time suggesting a similar level of tolerance to the extreme heat shock. However, changes at the cellular and biochemical level are usually the first detectable response to an environmental perturbation and can be observed before the physical signs of stress such as bleaching are evident (Bierkens, 2000). For this reason, during the progression of the coral bleaching, the modulation of the Hsp60 was analyzed. Hsp60 is an essential mitochondrial chaperone which is involved in numerous cellular processes including thermo protection (Choresh et al., 2001; Kregel, 2002). As shown in Table 1, the differences in Hsp60 modulation were significant both between and within species and time intervals. Starting from a similar basal constitutive level of Hsp60 under normal physiological conditions in all control samples, the immediate and extreme increase of water temperature to 34 C elicited a similar transient induction of Hsp60 expression for all the three coral species (Fig. 2). An initial strong significant up-regulation of Hsp60 already after the first 6 h of heat shock was observed (ANOVA, Tukey's HSD post hoc tests for pair-wise comparison of means; p < 0.05 compared with Time 0, Fig. 3), which is consistent with several studies indicating that the induction of Hsps response represents one of the first rapid defense cellular mechanisms (Robbart et al., 2004; Chow et al., 2009; Fitt et al., 2009; Seveso et al., 2013). Subsequently, in all the three species, a further increase of Hsp60 level took place reaching the highest values after 12 h of treatment (p < 0.05 compared with Time 0 and 6 h, Figs. 2 and 3). The gradual and intense increase of Hsp60 proceeded simultaneously with the increasing paleness of coral tissue (Fig. 1). Since it is known that even a small increase in temperature can cause protein unfolding and many of the morphological and phenotypic effects of heat stress can be explained by the aggregation of proteins and an imbalance of protein homeostasis in general
Table 1 Summary of the two-way analysis of variance (ANOVA) for all the normalized Hsp60 intensity values obtained from the different groups of samples. Species and exposure time were used as factors. Factors
Df
Sum sq
Mean sq
F Value
Pr(>F)
Species Time Species:Time Residuals
2 6 12 63
3.3698 26.2553 2.2081 0.6702
1.6849 4.3759 0.184 0.0106
158.381 411.331 17.297
0.05 compared with Time 0; p < 0.05 compared with 6 h). At the end of the treatment, Hsp60 is reduced below the basal level. Finally, in A. echinata the time profile of Hsp60 down-regulation was similar to that of M. monasteriata until 24 h of stress (Figs. 2C and 3). Afterwards, a strong and significant decrease in the Hsp60 levels took place and remained very low until the end of the exposure (Fig. 3). No significant difference was detected between the values recorded for M. monasteriata and A. echinata (p > 0.01, Wilcoxon signed-rank test), while the Hsp60 values recorded for S. hystrix at different times were significantly different from those recorded for both M. monasteriata and A. echinata (p < 0.01, Wilcoxon signed-rank test). It is necessary to mention that our results refer to the Hsp60 modulation of the coral polyp tissue since in our extracts Symbiodinium proteins have been removed. In fact, in S. hystrix no band, even after overexposure of the filter, was detected by the antiRubisco antibody which is specific to photosynthetic organisms (Fig. 4B). On the contrary, in line with the modulation reported in Fig. 2A, the anti-Hsp60 antibody detected the band relative to Hsp60 (Fig. 4C). In extracts prepared in parallel using glass beads (see Materials and Methods), the anti-Rubisco antibody detected an intense band in the control healthy sample at 27 C and in the sample subjected to 12 h of thermal stress. Interestingly, after 36 h of stress concomitant to the complete bleaching of the coral, no band was detected (Fig. 4B). Since in a coral endosymbiotic condition (Mayfield et al., 2009), it has been observed that the Rubisco
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Fig. 4. Western analysis of S. hystrix extracts. Samples from of healthy colonies (Time 0, 27 C) and colonies subjected to 12 and 36 h of heat shock (34 C) prepared by trituration with pestle and mortar followed (1) or not (2) by an additional cell disruption step with glass beads (see Methods for details) were subjected to Western analysis. The same amount of proteins was loaded on each lane. The filter was stained with Ponceau (A) and immunodecorated with anti-Rubisco antibody (B) and anti-Hsp60 antibody (C). The filter in B has been deliberately overexposed. MWM: pre-stained protein markers. RbcL: Rubisco protein standard. Representative filters are shown.
level was not affected by diel variation, remaining identical over one 12L:12D cycle (Mayfield et al., 2014), these data suggest that coral bleaching can be ascribed to the lost of zooxanthellae from the coral tissues. As expected, in the extracts prepared with glass beads as well, the anti-Hsp60 antibody detected Hsp60 but, at 27 C and
Fig. 3. Hsp60 levels of S. hystrix, M. monasteriata and A. echinata at different time exposure during the heat shock. The values were determined by densitometric analysis as described under Materials and Methods. Signals for 6 different blots for each species were analyzed. Data are expressed as arbitrary units and as mean ± SEM (two-way ANOVA followed by Tukey's HSD multiple pair-wise comparisons, *p < 0.05 compared to Time 0, #p < 0.05 compared to 6 h).
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in the sample subjected to 12 h of thermal stress, its level was higher compared to that of the extracts with no Symbiodinium contamination indicating that the antibody targets both polyps and Symbiodinium Hsp60, strengthening the signal (Fig. 4). Similar results were obtained for M. monasteriata and A. echinata (data not shown) confirming that the Hsp60 modulation we observed is not that of the coral holobiont. Taken together, our results are in line with the fact that Hsps represent a temporally dynamic but time-limited cellular machinery for combating the impacts of stress (Van Oppen and Gates, 2006). Furthermore, the down-regulation of the Hsp60 might occur when the environmental changes become physiologically intolerable for the organism and the defense mechanisms are no longer able to counteract the strong cellular stress (Carpenter et al., 2010; Seveso et al., 2013). In this context, the extreme conditions may result in increased damage and/or death of the organism due also to the energy expenditure which is required to neutralize or dissipate the effects of stress and restore cellular or tissue damage (Gates and Edmunds, 1999; Morgan and Snell, 2002). Moreover our results show that each species displayed different patterns of Hsp60 down-regulation after the 12 h of exposure which can be indicative of a different threshold of tolerance and resistance to the thermal stress. S. hystrix may represent the most susceptible species to heat stress, as the Hsp60 level quickly decreased and disappeared before the end of the treatment. On the contrary, M. monasteriata which displayed a more prolonged expression of Hsp60 might be the more tolerant and resistant species to thermal stress. In line with this, it has been reported that S. hystrix is among the first coral species to bleach and die during coral bleaching events and is widely considered a highly susceptible species with limited ability to tolerate increases in temperature over the average summer monthly mean: thus a “loser” organism in the face of environmental change (Hung et al., 1998; Marshall and Baird, 2000; Loya et al., 2001; van Woesik et al., 2011). This has been related to the physical properties of the corals, which influence gas and metabolite exchange across boundary layers affecting thermal susceptibility. Thin tissues and the low masstransfer efficiency in accordance with the branching morphology would make corals highly thermally susceptible due to the low photo-protective capacity of the tissues and the low ability to remove the accumulation of toxic oxygen radicals generated by the stresses (Lesser, 1997; Hoegh-Guldberg, 1999; Loya et al., 2001; Nakamura and van Woesik, 2001). In this context, S. hystrix is the coral species which displays the most finely branched morphology. On the contrary, the massive and especially plating corals, such as M. monasteriata, generally display thicker tissue and higher mass transfer than the branched species (Loya et al., 2001), allowing a greater resistance to environmental stress conditions and consequently making M. monasteriata less of a “loser”. Similarly, a different Hsp60 modulation over time among coral species which displayed different growth morphology was also observed for the sensitive branched coral Stylophora pistillata and the more resistant laminar coral Turbinaria reniformis subjected to heat shock (Chow et al., 2009). Finally, A. echinata displayed an intermediate response to thermal stress which could be generated by its branched morphology (but which is less fine than S. hystrix) and by evolutionary reasons as it belong to the same family of M. monasteriata (Acroporidae). In conclusion, our data suggest that the modulation of the Hsp60 could provide useful information about the susceptibility of the coral taxa to environmental disturbances. At the same time, these analyses might allow us to investigate in different types of coral post-stress recovery ability and resilience. Furthermore, it is essential to examine how other coral species assembling a coral community in a reef area respond differently to adverse
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Marine Environmental Research journal homepage: www.elsevier.com/locate/marenvrev
The susceptibility of corals to thermal stress by analyzing Hsp60 expression Davide Seveso a, b, *, Simone Montano a, b, Giovanni Strona c, Ivan Orlandi a, Paolo Galli a, b, Marina Vai a a b c
Department of Biotechnologies and Biosciences, University of Milan e Bicocca, Piazza della Scienza 2, 20126, Milan, Italy MaRHE Centre (Marine Research and High Education Centre), Magoodhoo Island, Faafu Atoll, Maldives European Commission, Joint Research Centre, Institute for Environment and Sustainability, Via E. Fermi 2749, I-21027 Ispra, Italy
a r t i c l e i n f o
a b s t r a c t
Article history: Received 21 March 2014 Received in revised form 23 May 2014 Accepted 13 June 2014 Available online 20 June 2014
Due to the increasing frequency and severity of the coral bleaching events in the context of global warming, there is an urgent need to improve our understanding of the susceptibility of corals to thermal stresses, particularly at the sub-cellular level. In this context, we examined the modulation of the polyp mitochondrial Hsp60 in three scleractinian coral species (Seriatopora hystrix, Montipora monasteriata and Acropora echinata) under simulated heat shock bleaching at 34 C during a time course of 36 h. All three species displayed a similar initial increase of Hsp60 level which accompanies the increasing paleness of coral tissue. Afterwards, each of them showed a specific pattern of Hsp60 down-regulation which can be indicative of a different threshold of resistance, although it proceeded in synchrony with the complete bleaching of tissues. The finely branched S. hystrix was the species most susceptible to heat stress while the plating M. monasteriata was the most tolerant one, as its Hsp60 down-regulation was less rapid than the branching corals. On the whole, the Hsp60 modulation appears useful for providing information about the susceptibility of the different coral taxa to environmental disturbances. © 2014 Elsevier Ltd. All rights reserved.
Keywords: Thermal susceptibility Coral bleaching Heat shock proteins Hsp60 Global climate change
1. Introduction Coral reefs worldwide are among the ecosystems most vulnerable to global climate change (Hughes et al., 2003; Veron et al., 2009). The primary response to hyperthermic stress is represented by coral bleaching events, caused by the breakdown in the relationship between scleractinian corals and their endosymbiotic dinoflagellates (genus Symbiodinium), which in recent decades have led to widespread coral mortality and reef degradation (Weis, 2008; Wilkinson, 2008). Projections about the sea temperature will soon overcome the well-known thermal thresholds for corals, and consequently bleaching events will increasingly become chronic, and affect the long-term viability of coral reefs (Donner et al., 2005; Hoegh-Guldberg et al., 2007). Given the threats of global climate change for coral reefs, there is an urgent need to develop proactive means and biomarkers capable of diagnosing the health of corals
* Corresponding author. Department of Biotechnologies and Biosciences, University of Milan e Bicocca, Piazza della Scienza 2, 20126, Milan, Italy. Tel.: þ39 0264483433. E-mail address: [email protected] (D. Seveso). http://dx.doi.org/10.1016/j.marenvres.2014.06.008 0141-1136/© 2014 Elsevier Ltd. All rights reserved.
before visible and irreparable bleaching takes place (Downs et al., 2000; Mayfield et al., 2011, 2013). The coral heat stress response involves a wide array of cellular and physiological processes, including heterotrophic plasticity, the production of protective pigments, mycosporine-like amino acids and the expression of fluorescent proteins and antioxidant enzymes (Richier et al., 2005; Grottoli et al., 2006; Weis, 2008; Baird et al., 2009). There is also evidence that corals have an enhanced thermotolerance capacity linked to cellular protective mechanisms such as the induction of the Heat shock proteins (Hsps), (Brown et al., 2002; Rosic et al., 2011; Chow et al., 2012). Hsps are ubiquitous molecular chaperones which regulate protein structure and function under physiological conditions, as well as during and after stresses (Hartl et al., 2011). In response to environmental stresses their expression is up-regulated in order to increase cellular repair and tolerance to adverse conditions by preserving metabolic functions (Richter et al., 2010). Among the Hsps, the mitochondrial 60-kDa chaperonin (Hsp60) is an important component of the folding system in the mitochondrial matrix and plays an essential role in both cellular physiology and stress response (Mayer, 2010). The increase in its level indicates a shift in the protein chaperoning and degradation within the mitochondria and implies a change in
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the mitochondrial-associated metabolic pathways (Papp et al., 2003). For these reasons, the expression of the Hsps, such as the Hsp60, can represent potentially useful molecular biomarkers for detecting early signs of change in an organism's physiological state, to the extent that in scleractinian corals the role of Hsp60 as a potential environment stress marker has been widely discussed (Brown et al., 2002; Downs et al., 2005; Rossi et al., 2006; Chow et al., 2009; Seveso et al., 2012, 2013). However, coral susceptibility to stress and bleaching appears to be highly variable since different corals display different levels of physiological resistance to environmental stress (Obura, 2001; McClanahan et al., 2007; Montano et al., 2010). The diversity among species in their susceptibility to disturbances represents a critical aspect of community dynamics, since it can give rise to changes in the community structure and species composition which determine the long-term persistence of coral reefs (Hughes and Connell, 1999). Attempts to understand the differences in the response of reef-building corals to thermal stresses have principally focused on coral morphology, growth and metabolic rates, tissue thickness and host CO2 supply strategies, with faster growing branching taxa being more severely affected by bleaching than slower growing massive taxa (Gates and Edmunds, 1999; Marshall and Baird, 2000; Loya et al., 2001; Darling et al., 2012; Wooldridge, 2014). Although there is an urgent need to understand the factors which determine this susceptibility, until now molecular mechanisms involved in coral thermal tolerance have not been completely elucidated (Weis, 2010), neither has the choice been made of a suitable and fast responding marker for the physiological stress response. In this regard, the use of Hsps as biomarkers to analyze the different susceptibility of reef-building corals to stresses has been partially investigated (Chow et al., 2009; Fitt et al., 2009). In this study we have examined and compared the Hsp60 expression profiles in three different species under simulated heat stress bleaching and during a time course of 36 h. In particular, these analyses were performed in 3 common reef-building scleractinian species belonging to Pocilloporidae (Seriatopora hystrix) and Acroporidae (Montipora monasteriata and Acropora echinata) families, which represent the most sensitive taxa to environmental stresses, since these corals typically showed similar thermal responses and suffered high mortality during bleaching events (Obura, 2005; Montano et al., 2010; Guest et al., 2012). 2. Materials and methods 2.1. Maintenance condition of the corals The coral colonies of S. hystrix, M. monasteriata and A. echinata (n ¼ 3 colonies per species) were maintained under laboratory culture conditions in a flow-through aquaria system at the Civic Aquarium of Milano (Italy) since 2011. The system is composed of 120-l tanks, connected to a 330-l sump containing gravel-bed filter, protein skimmer and a 500 W Titanium Heater (Aqua Medic) with a temperature controller. In all the tanks, the light was supplied by 400 W metal halide lamps (Powerstar HQI-T, Osram), which provided a constant photosynthetically active radiation (PAR) irradiance of 200 ± 30 mmol photons m2 s1 (mE), as reported by (Chow et al., 2009), measured by a photo-radiometer (Delta Ohm e HD2302.0). The photoperiod was set at 12 h:12 h light:dark cycle. Normal water temperature was set at 27 C (±0.2 C) and the 35 ppt of salinity was reproduced. Evaporation in aquaria was offset by the addition of deionized water. Calcium, magnesium, iodium and alkalinity were assessed and reintegrated every two days to ensure stable water chemistry. Salinity and pH of the seawater in aquarium tanks were constantly monitored using a Hach Lange HQ 30D flexi portable meter.
2.2. Experimental design and thermal stress experiment Four nubbins of similar large size were generated with pliers from each of the nine colonies for a total of thirty-six. They were randomly distributed into three aquaria chosen as experimental tanks in order to obtain four nubbins per species per aquarium which represented the biological replicates. For each species nine nubbins (3 per tank) were reserved for Western analyses and three nubbins (1 per tank) for morphological analyses. The coral fragments were maintained in the conditions described above for 10 days and left to recover from the handling procedure until tissue was found to have completely overgrown at the site of fracture (Mayfield et al., 2012). During the acclimatization period at 27 C all the chemical and physical parameters of the seawater were kept stable and constantly monitored. To minimize variations due to light bulb diffusion and water movement, the position of nubbins within each tank was changed every four days. Tanks were cleaned on a weekly basis in order to minimize algal growth (RodolfoMetalpa et al., 2005) and nubbins were visually inspected once a day to check for bleaching and mortality. After the acclimation period, the coral fragments of S. hystrix, M. monasteriata and A. echinata were photographed for morphological analyses and the others were sampled for Western analyses just clipping off a small piece of skeleton with bone-cutting pliers. They represented the control samples (Time 0). After sampling, coral fragments were immediately frozen in liquid nitrogen and stored at 80 C until analysis. Only healthy coral fragments exhibiting healing at fragmentation lesions, growth and dark pigmentation were used as control and for subsequent experiments. Afterwards, all the nubbins of S. hystrix, M. monasteriata and A. echinata were subjected to severe and extreme heat shock at 34 C (±0.2 C) with the aim of inducing coral bleaching and a subcellular thermal stress response at the end of the treatment. For six of the nine nubbins per species reserved for Western analyses, the exposures were carried out for 36 h and the sampling were performed 6, 12, 18, 24, 30 and 36 h after 34 C have been reached. All the samples were immediately frozen in liquid nitrogen and stored at 80 C prior to protein extraction. As the same 18 nubbins were sampled six times, the assumption was made that sampling of a portion of the colony representing less than 0.1% of the total colony volume would not cause stress to another section of the same colony (Mayfield et al., 2011). Furthermore, as artificial lighting was used during the experiments, sampling was performed at the same light level with the exception of the sampling performed after 18 h which was realized during the dark period. After 12 and 36 h at 34 C, a photographic documentation of coral colonies was performed in order to monitor the coral health as tissue coloration and morphological aspect. Moreover, the remaining three nubbins per species reserved for Western analyses after 18 h of thermal stress were returned to 27 C. The sampling of these coral fragments were performed after 12 h and at the end of the recovery treatment. Temperature, PAR, pH, salinity and the other chemical parameters were checked routinely over the course of the experiment. During stress exposures each aquarium tank represented a closed system (static flow) containing Koralia immersed pumps (1200 l/h) in order to continuously mix and aerate the seawater. Elevated temperature was achieved using submersible Acqua Medic Titanium Aquarium Heaters (model TH-100, 240 V) connected to a temperature controller (Eliwell, PC 902T). 2.3. Protein extracts and Western analysis In the laboratory, the frozen coral fragments were powdered using mortar and pestle. Extracts containing only polyp proteins were prepared by homogenization in SDS-buffer, boiling and
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centrifugation steps as previously described (Seveso et al., 2013), in order to remove any Symbiodinium contamination. Moreover, before boiling, for some extracts an equal volume of acid-washed glass beads (425e600 mm, Sigma) was added followed by vigorously shaking on a vortex (10 cycles of 1 min) interspersed with cooling on ice. This latter procedure has been used to prepare extracts containing also Symbiodinium proteins (Weis et al., 2002; Seveso et al., 2013). All the extracts were frozen at 20 C until used. Aliquots were used for protein concentration determinations using the Bio-Rad protein assay kit (Bio-Rad Laboratories). Protein samples were separated by SDS-PAGE on 8% polyacrylamide gels (Vai et al., 1986) and the same amount of proteins was loaded on each lane of the gel. Duplicate gels were run in parallel and after electrophoresis one gel was stained with Coomassie Brilliant Blue to visualize the total proteins and the other electroblotted onto nitrocellulose membrane for Western analysis (Seveso et al., 2012). Correct protein transfer was confirmed by Ponceau S Red (SigmaeAldrich) staining of filters. For each blot, the same amount of recombinant human Hsp60 (Enzo Life Sciences) was included as an internal control for signal differences across blots. Immunodecoration with anti-Hsp60 monoclonal antibody (IgG mouse clone LK-2, SPA-807, Enzo Life Sciences), anti-b-Actin monoclonal antibody (clone C4, MAB1501, Millipore) and secondary antibody anti-mouse IgG conjugated with horseradish peroxidase (Thermo Scientific) was performed as previously reported (Seveso et al., 2013). To evaluate the presence/absence of Symbiodinium proteins, filters were probed with anti-Ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) large subunit antibody (AS03037, Agrisera) followed by incubation with secondary antibody anti-rabbit IgG conjugated with
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horseradish peroxidase (Thermo Scientific) as described (Seveso et al., 2013). Rubisco protein standard (AS01017S, Agrisera) was loaded as internal control. Binding was visualized with the Pierce ECL Western Blotting Substrate followed by X-ray films. 2.4. Densitometric and statistical analyses Densitometric analysis was performed on a Bio-Rad GS-800 calibrated imaging densitometer and quantified with the Image J free software (http://rsb.info.nih.gov/ij/) of the NIH Image software package (National Institutes of Health, Bethesda, Md.). For each blot, the scanned intensity of the Hsp60 bands was normalized against the intensity of the b-Actin ones. Although the housekeeping gene encoding b-Actin has been shown to undergo diel fluctuations in its expression in corals (Mayfield et al., 2010), in all our experiments the b-Actin level did not display a significant modulation under the different exposure time and for each species. Data were expressed as the mean ± standard error of the mean (SEM). Data normality was verified using ShapiroeWilk test. Twoway analysis of variance (ANOVA) was then performed for all the normalized Hsp60 intensity values obtained from the different groups of samples, using species and exposure time as factors. Following two-way ANOVA, a Tukey's HSD post hoc test was performed to assess significant differences (p < 0.05) in Hsp60 protein levels in response to the different exposure times for each species. A non-parametric paired t-test (Wilcoxon signed-rank test) was performed to assess if Hsp60 expression at different times was significantly different in each pair of species.
Fig. 1. Effect of heat shock treatment on coral colonies. Representative photographic documentation of the morphological characteristics of S. hystrix (A), M. monasteriata (B) and A. echinata (C) colonies at 27 C (control, Time 0) and after 12 and 36 h of heat shock at 34 C (scale bar: 1 cm).
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3. Results and discussion Climate change has led to an increase of studies regarding the mechanisms of the stress response at sub-cellular level, especially those that may confer enhanced tolerance to changing environmental conditions for sessile marine taxa, such as scleractinian corals, that cannot easily migrate to new environmental optima (Mayfield and Gates, 2007; Somero, 2012; Doney et al., 2012). In particular, here we focused on the heat shock responses among different coral species during a bleaching event by analyzing and comparing their Hsp60 patterns of expression, with the aim of elucidating the role of Hsps in coral resistance and susceptibility to thermal stresses. To do this, colonies of S. hystrix, M. monasteriata and A. echinata were subjected to an extreme temperature of 34 C for 36 h, starting from a control temperature of 27 C. At first, as expected, morphological analyses of the corals indicated that all the colonies belonging to the three species suffered intense bleaching. In fact, already after 12 h of heat shock all the colonies displayed retracted polyps and pale tissues which became completely bleached at the end of the exposure (Fig. 1). Despite the dissimilar coral growth forms, no significant difference in tissue appearance at the various exposure time was observed among species, which displayed a similar bleaching trend over time suggesting a similar level of tolerance to the extreme heat shock. However, changes at the cellular and biochemical level are usually the first detectable response to an environmental perturbation and can be observed before the physical signs of stress such as bleaching are evident (Bierkens, 2000). For this reason, during the progression of the coral bleaching, the modulation of the Hsp60 was analyzed. Hsp60 is an essential mitochondrial chaperone which is involved in numerous cellular processes including thermo protection (Choresh et al., 2001; Kregel, 2002). As shown in Table 1, the differences in Hsp60 modulation were significant both between and within species and time intervals. Starting from a similar basal constitutive level of Hsp60 under normal physiological conditions in all control samples, the immediate and extreme increase of water temperature to 34 C elicited a similar transient induction of Hsp60 expression for all the three coral species (Fig. 2). An initial strong significant up-regulation of Hsp60 already after the first 6 h of heat shock was observed (ANOVA, Tukey's HSD post hoc tests for pair-wise comparison of means; p < 0.05 compared with Time 0, Fig. 3), which is consistent with several studies indicating that the induction of Hsps response represents one of the first rapid defense cellular mechanisms (Robbart et al., 2004; Chow et al., 2009; Fitt et al., 2009; Seveso et al., 2013). Subsequently, in all the three species, a further increase of Hsp60 level took place reaching the highest values after 12 h of treatment (p < 0.05 compared with Time 0 and 6 h, Figs. 2 and 3). The gradual and intense increase of Hsp60 proceeded simultaneously with the increasing paleness of coral tissue (Fig. 1). Since it is known that even a small increase in temperature can cause protein unfolding and many of the morphological and phenotypic effects of heat stress can be explained by the aggregation of proteins and an imbalance of protein homeostasis in general
Table 1 Summary of the two-way analysis of variance (ANOVA) for all the normalized Hsp60 intensity values obtained from the different groups of samples. Species and exposure time were used as factors. Factors
Df
Sum sq
Mean sq
F Value
Pr(>F)
Species Time Species:Time Residuals
2 6 12 63
3.3698 26.2553 2.2081 0.6702
1.6849 4.3759 0.184 0.0106
158.381 411.331 17.297
0.05 compared with Time 0; p < 0.05 compared with 6 h). At the end of the treatment, Hsp60 is reduced below the basal level. Finally, in A. echinata the time profile of Hsp60 down-regulation was similar to that of M. monasteriata until 24 h of stress (Figs. 2C and 3). Afterwards, a strong and significant decrease in the Hsp60 levels took place and remained very low until the end of the exposure (Fig. 3). No significant difference was detected between the values recorded for M. monasteriata and A. echinata (p > 0.01, Wilcoxon signed-rank test), while the Hsp60 values recorded for S. hystrix at different times were significantly different from those recorded for both M. monasteriata and A. echinata (p < 0.01, Wilcoxon signed-rank test). It is necessary to mention that our results refer to the Hsp60 modulation of the coral polyp tissue since in our extracts Symbiodinium proteins have been removed. In fact, in S. hystrix no band, even after overexposure of the filter, was detected by the antiRubisco antibody which is specific to photosynthetic organisms (Fig. 4B). On the contrary, in line with the modulation reported in Fig. 2A, the anti-Hsp60 antibody detected the band relative to Hsp60 (Fig. 4C). In extracts prepared in parallel using glass beads (see Materials and Methods), the anti-Rubisco antibody detected an intense band in the control healthy sample at 27 C and in the sample subjected to 12 h of thermal stress. Interestingly, after 36 h of stress concomitant to the complete bleaching of the coral, no band was detected (Fig. 4B). Since in a coral endosymbiotic condition (Mayfield et al., 2009), it has been observed that the Rubisco
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Fig. 4. Western analysis of S. hystrix extracts. Samples from of healthy colonies (Time 0, 27 C) and colonies subjected to 12 and 36 h of heat shock (34 C) prepared by trituration with pestle and mortar followed (1) or not (2) by an additional cell disruption step with glass beads (see Methods for details) were subjected to Western analysis. The same amount of proteins was loaded on each lane. The filter was stained with Ponceau (A) and immunodecorated with anti-Rubisco antibody (B) and anti-Hsp60 antibody (C). The filter in B has been deliberately overexposed. MWM: pre-stained protein markers. RbcL: Rubisco protein standard. Representative filters are shown.
level was not affected by diel variation, remaining identical over one 12L:12D cycle (Mayfield et al., 2014), these data suggest that coral bleaching can be ascribed to the lost of zooxanthellae from the coral tissues. As expected, in the extracts prepared with glass beads as well, the anti-Hsp60 antibody detected Hsp60 but, at 27 C and
Fig. 3. Hsp60 levels of S. hystrix, M. monasteriata and A. echinata at different time exposure during the heat shock. The values were determined by densitometric analysis as described under Materials and Methods. Signals for 6 different blots for each species were analyzed. Data are expressed as arbitrary units and as mean ± SEM (two-way ANOVA followed by Tukey's HSD multiple pair-wise comparisons, *p < 0.05 compared to Time 0, #p < 0.05 compared to 6 h).
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in the sample subjected to 12 h of thermal stress, its level was higher compared to that of the extracts with no Symbiodinium contamination indicating that the antibody targets both polyps and Symbiodinium Hsp60, strengthening the signal (Fig. 4). Similar results were obtained for M. monasteriata and A. echinata (data not shown) confirming that the Hsp60 modulation we observed is not that of the coral holobiont. Taken together, our results are in line with the fact that Hsps represent a temporally dynamic but time-limited cellular machinery for combating the impacts of stress (Van Oppen and Gates, 2006). Furthermore, the down-regulation of the Hsp60 might occur when the environmental changes become physiologically intolerable for the organism and the defense mechanisms are no longer able to counteract the strong cellular stress (Carpenter et al., 2010; Seveso et al., 2013). In this context, the extreme conditions may result in increased damage and/or death of the organism due also to the energy expenditure which is required to neutralize or dissipate the effects of stress and restore cellular or tissue damage (Gates and Edmunds, 1999; Morgan and Snell, 2002). Moreover our results show that each species displayed different patterns of Hsp60 down-regulation after the 12 h of exposure which can be indicative of a different threshold of tolerance and resistance to the thermal stress. S. hystrix may represent the most susceptible species to heat stress, as the Hsp60 level quickly decreased and disappeared before the end of the treatment. On the contrary, M. monasteriata which displayed a more prolonged expression of Hsp60 might be the more tolerant and resistant species to thermal stress. In line with this, it has been reported that S. hystrix is among the first coral species to bleach and die during coral bleaching events and is widely considered a highly susceptible species with limited ability to tolerate increases in temperature over the average summer monthly mean: thus a “loser” organism in the face of environmental change (Hung et al., 1998; Marshall and Baird, 2000; Loya et al., 2001; van Woesik et al., 2011). This has been related to the physical properties of the corals, which influence gas and metabolite exchange across boundary layers affecting thermal susceptibility. Thin tissues and the low masstransfer efficiency in accordance with the branching morphology would make corals highly thermally susceptible due to the low photo-protective capacity of the tissues and the low ability to remove the accumulation of toxic oxygen radicals generated by the stresses (Lesser, 1997; Hoegh-Guldberg, 1999; Loya et al., 2001; Nakamura and van Woesik, 2001). In this context, S. hystrix is the coral species which displays the most finely branched morphology. On the contrary, the massive and especially plating corals, such as M. monasteriata, generally display thicker tissue and higher mass transfer than the branched species (Loya et al., 2001), allowing a greater resistance to environmental stress conditions and consequently making M. monasteriata less of a “loser”. Similarly, a different Hsp60 modulation over time among coral species which displayed different growth morphology was also observed for the sensitive branched coral Stylophora pistillata and the more resistant laminar coral Turbinaria reniformis subjected to heat shock (Chow et al., 2009). Finally, A. echinata displayed an intermediate response to thermal stress which could be generated by its branched morphology (but which is less fine than S. hystrix) and by evolutionary reasons as it belong to the same family of M. monasteriata (Acroporidae). In conclusion, our data suggest that the modulation of the Hsp60 could provide useful information about the susceptibility of the coral taxa to environmental disturbances. At the same time, these analyses might allow us to investigate in different types of coral post-stress recovery ability and resilience. Furthermore, it is essential to examine how other coral species assembling a coral community in a reef area respond differently to adverse
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