Oxygen consumption in Mediterranean octocorals under different temperatures

Journal of Experimental Marine Biology and Ecology 390 (2010) 39–48

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Journal of Experimental Marine Biology and Ecology j o u r n a l h o m e p a g e : w w w. e l s ev i e r. c o m / l o c a t e / j e m b e

Oxygen consumption in Mediterranean octocorals under different temperatures Monica Previati a,b,⁎, Alice Scinto b, Carlo Cerrano b, Ronald Osinga a a b

AFI (Aquaculture and Fisheries Group), Animal Sciences Department, Wageningen University Marijkeweg 40, 6709 PG Wageningen, The Netherlands Dip.Te.Ris. (Dipartimento per lo Studio del Territorio e delle sue Risorse), Università di Genova, Corso Europa 26, 16132 Genova, Italy

a r t i c l e

i n f o

Article history: Received 2 April 2009 Received in revised form 14 April 2010 Accepted 15 April 2010 Keywords: Anthozoa Alcyonaria Respiration Polyp rhythm Necrosis

a b s t r a c t Ecosystem resilience to climate anomalies is related to the physiological plasticity of organisms. To characterize the physiological response of some common Mediterranean gorgonians to fluctuations in temperature, four species (Paramuricea clavata, Eunicella singularis, Eunicella cavolinii and Corallium rubrum) were maintained in aquaria, in which the temperature was increased every ten days with increments of 2– 3 °C, starting at 14 °C, ending at 25 °C. Oxygen consumption, number of open/closed polyps and percentage of necrotic tissue were monitored. All species showed similar activity patterns with increasing temperature. P. clavata and E. singularis showed the highest respiration rate at 18 °C, E. cavolinii and C. rubrum at 20 °C. Above these temperatures, both oxygen consumption and polyp reactivity decreased in all species. The present data confirm a reduction of the metabolic activity in Mediterranean gorgonians during periods of high temperature. At temperatures above 18 °C, the percentage of open polyps (considered as a parameter to evaluate polyps reactivity) decreased, thus mirroring the trend of oxygen consumption. The average values of Q10 indicated that gorgonians have a definite temperature limit over which the metabolism (oxygen consumption) stop to follow the temperature increase. After three days at 25 °C, metabolic activity in E. cavolinii, C. rubrum and P. clavata further decreased and the first signs of necrosis were observed. At this temperature, activity remained unchanged in E. singularis. This species seems to more resistant to thermal stress. The symbiotic zooxanthellae present in this species are likely to provide an alternative source of energy when polyps reduce their feeding activity. © 2010 Elsevier B.V. All rights reserved.

1. Introduction In the Mediterranean Sea, many abiotic factors fluctuate throughout the year. Due to seasonal patterns, annual changes in temperature in the Mediterranean have a range of up to 15 °C (Founda et al., 2004). Such strong fluctuations in temperature have profound impact on animal metabolism. Temperature and metabolism have been related since the work of Arrhenius (1889; 1915) and Van't Hoff (1896), who proposed a mechanistic approach. However, due to the complexity of cellular life and the metabolic feedback mechanisms by which cells respond to temperature fluctuations, the thermodynamic response to temperature cannot be predicted from physical principles only (Clarke and Fraser, 2004). Thermo-tolerance is likely to be settled by the adjustment of the oxygen demand and influences the metabolic activities and energy-demanding processes in cells. This is particularly the case for benthic sessile organisms (Arillo et al., 1989; Frederich and Pörtner, 2000; Hochachka and Somero, 2002; Clarke, 2003; Hadas et al., 2008), which cannot easily migrate to other, more suitable habitats. ⁎ Corresponding author. Dip.Te.Ris. (Dipartimento per lo Studio del Territorio e delle sue Risorse) Università di Genova, Corso Europa 26, 16132 Genova, Italy. Tel.: +39 010 3538563; fax: +39 010 3538220. E-mail address: [email protected] (M. Previati). 0022-0981/$ – see front matter © 2010 Elsevier B.V. All rights reserved. doi:10.1016/j.jembe.2010.04.025

Tolerance to naturally occurring fluctuations is likely to be challenged by global warming (Gambiani et al., 2009). Due to global change, the upper threshold for temperature tolerance may be exceeded, which will have profound impact on marine life, in particular on benthic communities. As reviewed by Hughes (2000), recent climatic and atmospheric trends are already affecting species physiology, distribution and phenology. In the NW Mediterranean Sea, massive mortality events are occurring nearly every summer/ autumn season, involving mainly benthic invertebrates between 0 and 50 m depth (Cerrano et al., 2000; Perez et al., 2000; Garrabou et al., 2001; Cerrano et al., 2005; Linares et al., 2005; Garrabou et al., 2009). These episodes may be due to synergistic effects of thermal anomalies, summer energy shortage, and pathogens (Romano et al., 2000; Garrabou et al., 2001; Lesser et al., 2007; Cerrano and Bavestrello, 2008; Coma et al., 2009; Ferrier-Pagès et al., 2009; Vezzulli et al., 2010). Moreover, high temperatures reduce the oxygen solubility (Carpenter, 1966; Truesdale et al., 1955), thus limiting its availability. The importance of oxygen availability for marine invertebrates has recently been summarized by Riedel et al. (2008), evidencing both direct (physiological) and indirect (behavioural) effects. A reduced availability of oxygen coinciding with an increased metabolic oxygen demand may cause severe oxygen limitation in marine invertebrates under high temperatures (Legovic and Justic, 1997). In this context, it is important to describe patterns in

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respiration activity of marine invertebrates under increasing temperature, in particular for those species that are most vulnerable to climate anomalies. Such knowledge is needed to draw a more complete scenario of the parameters possibly involved in mass mortalities. Gorgonians (Cnidaria, Alcyonaria) are among the most endangered groups by mass mortality events (Cerrano and Bavestrello, 2008; Garrabou et al., 2009). Sea-fans, a typical component of the benthic Mediterranean communities, are considered important foundation species of coralligenous habitats, amplifying the ecosystem complexity from both a physical and biological point of view (Sarà, 1969; True, 1970; Gili and Ros, 1985; Sebens, 1991; Ballesteros, 2006; Scinto et al., 2009). Since the last ten years, these ecosystem engineer species suffered from mass mortality with a biomass decrease of more than 50% in the case of Paramuricea clavata (Linares et al., 2005). Despite the large number of studies about mass mortality events (see Cerrano and Bavestrello, 2008 for a review), gorgonian physiology is still poorly known. Some data are available for the symbiotic with zooxanthellae Eunicella singularis where oxygen consumption is clearly affected by light exposure (Brafield et al., 1965; Chapman and Theodor, 1969; Ferrier-Pagès et al., 2009). Concerning the oxygen consumption of P. clavata, the only data available were collected in situ in the Mediterranean Sea by Coma et al. (2002), who found that low respiration rates during summer correlated with a low availability of planktonic food. The aim of this work is to measure the effects of temperature increase on the physiology of four of the most abundant octocorals species of the Mediterranean pre-coralligenous and coralligenous communities by, analyzing oxygen consumption, polyps activity and necrosis signs of these octocorals under controlled aquarium conditions. 2. Materials and methods 2.1. The species studied and their habitats To facilitate the interpretation of results it is important to take in account some ecological peculiarities of the four studied species and their habitat. The coralligenous communities are among the most important hot-spot of biodiversity in the Mediterranean Sea. In terms of biomass, Cnidaria are the main constituent of these communities (see Ballesteros, 2006 for a review). True (1970) described three different coralligenous assemblages: one dominated by Eunicella cavolinii (Von Koch, 1887), one dominated by P. clavata (Risso, 1826) and one dominated by Corallium rubrum (Linnaeus, 1758). E. cavolinii has a bathymetric distribution that ranges from 10 to 150 m, with the optimum between 15 and 70 m depth. It preferably grows on vertical substrates, orienting itself to the main currents. As such, it is regarded an indicator of the prevailing water flow (Russo, 1985). Although E. cavolinii is a common species, there is little information regarding its ecology. It can live sympatrically with P. clavata suggesting similar ecological needs. The red gorgonian (P. clavata) is considered a true ecosystem engineering species, because its presence strongly affects community structure, both in terms of biomass and in terms of biodiversity (Scinto et al., 2009). Its depth distribution ranges from 5 to 110 m (Weinberg, 1991) with an optimum between 35 and 80 m depth. This species has an evident skiophilous habitus and needs high water movement (True, 1970; Weinberg, 1978; Mistri, 1994; Linares et al., 2007). C. rubrum has the widest bathymetric distribution, ranging from 7 to 200 m, with the optimum between 30 and 100 m depth (Giannini et al., 2003; Tsounis et al., 2006). The lack of sedimentation, dim-light or dark conditions, and a continuous but not intense water movement

are the main ecological requirements for this species. Among the four species studied, C. rubrum is the only one with dimorphic polyps (autozooids and siphonozooids). E. singularis (Esper, 1794), is the only Mediterranean gorgonian that lives symbiotically with unicellular algae (zooxanthellae), shows a bathymetric range comprised between 5 and 70 m depth (Weinberg and Weinberg, 1979; Weinberg, 1991). It is predominantly present on horizontal substrata (both hard and detritic) and it is a common species in pre-coralligenous assemblages (Linares et al., 2008). 2.2. Sampling and aquarium maintenance During winter 2008 nine apical fragments (about 5 cm long) of each of the four species studied were randomly collected between 20 and 30 m depth at the Marine Protected Area of Portofino (Ligurian Sea, Italy). Fragments were cut from the primary branch of each specimen sampled (one fragment per specimen). Directly underwater, each fragment was placed in plastic 50 ml tubes that were then transferred within 24 h to the facilities of the Aquaculture and Fisheries Group at Wageningen University, The Netherlands. During transfer, fragments were kept at 14 °C, the same temperature of the natural environment during collection. In order to acclimate the fragments, they were kept in an aquarium (70 cm long × 34 cm deep × 25 cm wide) for two weeks under stable conditions (temperature: 14 °C, salinity: 37.7–38‰, irradiance by artificial light: 5.8–5.5 µE m− 2 s− 1 for 12 h following a day/night rhythm) before starting the temperature experiments. Water flow in the aquarium was generated by two pumps: the first pump re-circulated the water through a temperature controller (1/ 5HP TECO SeaChill Chiller TR15 ± 0.5 °C) to maintain a constant temperature, while the second pump (located in the tank) created a constant, turbulent water movement (0.9 ± 0.1 cm/s) around the colonies. The aquarium water was continuously filtered through activated carbon filters and oxygenated with an air stone, which was positioned at the top of the aquarium in order to minimize its influence on the polyp activity. The tank was cleaned regularly to remove any microalgal build up. The gorgonians were fed every day with 1 ml of concentrated baby brine shrimp (Artemia nauplii) suspension. 2.3. Temperature regimes After two weeks of acclimation, the temperature in the aquarium was raised every ten days with 2 °C–3 °C increments: 14 °C (this initial temperature was maintained for another 10 days consecutive to the 14-day acclimation period), 16 °C, 18 °C, 20 °C, 22 °C and 25 °C. The temperatures between 14 °C and 22 °C match the average seasonal temperatures measured between 15 and 30 m depth in the studied area during sampling dives using a UWATEC underwater thermometer (±0.5 °C). According to a previous study (Bally and Garrabou, 2007), the 25 °C value represents the upper thermal tolerance limit, especially for P. clavata. After each temperature increase, the gorgonians were allowed to acclimate for five days, while the other five days were used to perform oxygen consumption measurements. The highest temperature regime (25 °C) was maintained only for one week and the oxygen consumption was measured on the first and third day. 2.4. Oxygen consumption To analyze the oxygen consumption under different temperature regimes, three fragments of each species were separately put in hermetic glass chambers. The cap of the chamber bears a hole where a luminescence sensor (HACH LDO® Process Dissolved Oxygen Probe) is inserted to monitor the oxygen concentration (HQ40d Dual-Input Multi-Parameter Digital Meter) in the chamber. The measured

M. Previati et al. / Journal of Experimental Marine Biology and Ecology 390 (2010) 39–48

magnitude of luminescent intensity and/or average relaxation time (luminescence lifetime) is inversely proportional to the concentration of oxygen and thus, the signal to noise ratio of the measurement increases with decreasing DO (dissolved oxygen) concentration. To avoid oxygen production by phototrophic symbionts, the experiments were performed under nearly dark conditions (irradiance 2–1.7 µE m− 2 s− 1). The oxygen concentration and the temperature were monitored every 5 min for 8 h. Each fragment was transferred into the chamber 1 h before starting the respiration measurements, in order to allow maximal expansion of the polyps. During each measurement, one empty chamber was used as control. The oxygen concentration value of control chambers has been subtracted from the value of respiration rates measured in the sample chamber. The water in the chambers was constantly mixed by a magnetic stirrer. Experiments carried out in the field (Coma et al., 2002) showed that a decrease in the oxygen concentration up to 15% neither affected the behaviour of the colonies, nor their respiration rates. For this reason, measurements were always stopped if the oxygen concentration inside the chamber dropped below 85% of the initial value. At the end of the experimental cycle, the dry mass (DM — drying at 90 °C for 24 h) and the ash free dry mass (AFDM — combusting at 450 °C for 5 h) were determined in order to estimate the organic content of the samples (Ribes et al., 2000). The samples were weighed using a Mettler AT 200 analytical balance. To examine the temperature dependence of the respiration rate, the value of Q10 has been calculated using the following equation that considers both the time of the incubation and the temperature: h i10 = T −T 2 1 t =8 Q10 = ∑ ðK2t 1−K1t 1Þ=t

where K1 represents the respiration rate at temperature T1, K2 the respiration at temperature T2 and t the duration of the oxygen measurement. 2.5. Polyp activity The polyp activity was analysed daily, observing each fragment before and after food supply. Following the approach (slightly modified) of Torrents et al. (2008), two different modes of activity were distinguished: expanded (this category included semi-expanded polyps, too) and retracted polyps. The percentage of open polyps was then calculated as: number of open polyps / total number of polyps × 100%. 2.6. Necrosis pattern The occurrence of necrosis phenomena on the gorgonian fragments was visually detected as: 1) the change of the coenenchyme colour; 2) the appearance of denuded axis. Fragments were photographed the first and the third day after increasing the temperature to the highest level of 25 °C. The pictures were analyzed by Image-J 1.28 free software. When observed, the percentage of necrotic tissue was recorded. 2.7. Statistical analyses Two-way ANOVA and subsequent Bonferroni's post-hoc testing were used to compare, for each species tested, differences in respiration rates between: 1) different temperatures (14 °C, 16 °C, 18 °C, 20 °C, 22 °C and 25 °C), and 2) different incubation intervals (between 0 and 480 min). The same approach was used to compare differences in the percentage of open polyps under different temperature and feeding conditions (i.e. before and after feeding). The relationship between oxygen consumption and polyp activity

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under different temperature regimes was calculated by standard correlation analysis (Pearson's correlation). For all statistical analyses, the SPSS 15.0, 2008 software package was used.

3. Results 3.1. Oxygen consumption Both the oxygen concentration and the temperature in all control measurements did not change significantly during the 8 h of incubation. In all experiments, the initial oxygen concentration was saturating, the concentration being in line with expected saturation values for the temperatures applied, ranging from 7.89 ± 0.17 mg O2 l− 1 at 14 °C to 6.95± 0.22 mg O2 l− 1 at 25 °C (Fig. 1). The oxygen consumption rates of the four tested gorgonians differed significantly under different temperature regimes (ANOVA, P b 0.000, Table 1). When plotting oxygen consumption rates against temperature (Fig. 1), all species exhibited a similar pattern with the highest respiration rates occurring at 18 °C to 20 °C. Despite the similarity in the overall patterns, small differences were found between species (ANOVA, P b 0.005). The respiration rate of C. rubrum started to increase at 18 °C, with a peak on 20 °C (0.92 ± 0.12 mg O2 g AFDM− 1 h− 1) (means ± SD), and a fast decrease at 25 °C (0.2 ± 0.14 mg O2 g AFDM− 1 h− 1) (means ± SD). After three days at high temperature (25 °C) C. rubrum showed the lowest respiration rate in comparison with the other species (0.08 ± 0.01 mg O2 g AFDM− 1 h− 1) (Table 2, Fig. 1A). P. clavata showed a bell-shaped trend between 14 °C and 22 °C with a peak at 18 °C (0.91 ± 0.22 mg O2 g AFDM− 1 h− 1) (means ± SD) and a decrement at 25 °C, both on the first and third day (0.26 ± 0.1 mg O2 g AFDM− 1 h− 1 and 0.09 ± 0.07 mg O2 g AFDM− 1 h− 1 on the first and third day at 25 °C, respectively) (Table 2, Fig. 1B). E. cavolinii showed a different pattern of oxygen consumption, with an increase at 16 °C, a slight decrease at 18 °C, a peak at 20 °C (0.61 ± 0.16 mg O2 g AFDM− 1 h− 1), a decrease at 22 °C and the lowest oxygen consumption after three days of high and prolonged temperature (25 °C) (0.12 ± 0.08 mg O2 g AFDM− 1 h− 1) (means ± SD) (Table 2, Fig. 1C). The oxygen consumption of E. singularis showed a bell-shaped trend from 14 °C to 22 °C with a peak at 18 °C (0.66 ± 0.09 mg O2 g AFDM− 1 h− 1) (means ± SD). A slight increase at 25 °C was detected, both at the first and third day (0.21 ± 0.09 mg O2 g AFDM− 1 h− 1 and 0.23 ± 0.11 mg O2 g AFDM− 1 h− 1 the first and third day, respectively) (means ± SD) (Table 2, Fig. 1D). The respiration rate was not constant during the incubation period (Table 1). When plotting oxygen consumption rate against time (Fig. 2), all species showed significant differences between incubation intervals (ANOVA, P b 0.000). C. rubrum showed a significantly increase of oxygen consumption after the first and third hour and a subsequent constant consumption when incubated at 14 °C and 16 °C. At 18 °C, the oxygen consumption increased until the fourth hour. At 20 °C, 22 °C and 25 °C (Day 3), the oxygen consumption of C. rubrum increased only after the first hour and then remained constant (Fig. 2A). After one day at 25 °C, a different pattern was observed. P. clavata showed an increase of oxygen consumption until the third hour when incubated at 14 °C, 20 °C and 22 °C. At 16 and 18 °C, the respiration rate continued to increases until the sixth hour. At 25 °C, the respiration rate increased only after the first hour and then decreased (Fig. 2B). At 14 °C, 16 °C, 18 °C, 20 °C and 22 °C, E. cavolinii showed a high oxygen consumption during the first and the second hour. During the third hour, the oxygen consumption decreased and remained constant until the end of the incubation. At 25 °C, the oxygen

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Fig. 1. Oxygen consumption rate (mg O2 g AFDM− 1 h− 1) (average ± ES) of the four Mediterranean species under different temperature regimes (between 14 °C and 25 °C). The oxygen consumption was analyzed after ten days incubation for the temperature range 14 °C–22 °C and on the first and the third day at 25 °C. (A) = Corallium rubrum; (B) = Paramuricea clavata; (C) = Eunicella cavolinii; (D) = Eunicella singularis.

consumption increased only after the first hour, after which it decreased continuously up to the end of the incubation (Fig. 2C). E. singularis showed a maximum oxygen consumption always at the second hour of the incubation, followed by a gradual decrease until the end of the incubation period (Fig. 2D). The average values of Q10 vary from 1.02 for C. rubrum to 1.4 for P. clavata. For E. cavolinii and E. singularis the values are 1.34 and 1.25,

respectively. Separate calculation of the Q10 values for different temperature ranges shows a different picture. In the range from 14 °C to 20 °C the Q10 values are above 1 for all species (1.56, 1.9, 1.8, 1.64 to C. rubrum, P. clavata, E. cavolinii and E. singularis, respectively), whereas between 22 °C and 25 °C the values of Q10 decrease below 1 (0.48, 0.87, 0.89, 0.85 in C. rubrum, P. clavata, E. cavolinii and E. singularis, respectively).

M. Previati et al. / Journal of Experimental Marine Biology and Ecology 390 (2010) 39–48 Table 1 Analysis of variance (repeated measures) for respiration rate on different temperature (between 14 °C and 25 °C) and in the different time (SS = Sum of Squares; *** = 0.001 b p; ** = 0.001 b p ≤ 0.01). Source

SS

df

Mean square

Species Error Temperature Temperature ⁎ Species Error (Temperature) Time Time ⁎ Species Error (Time) Temperature ⁎ Time Temperature ⁎ Time ⁎ Species Error (Temperature ⁎ Time)

1.650 0.224 10.772 10.621 1.080 9.643 4.240 0.369 6.283 8.844 1.718

3 8 6 18 48 8 24 64 48 144 384

0.550 0.028 1.795 0.590 0.022 1.205 0.177 0.006 0.131 0.061 0.004

F

p 19.651

***

79.800 26.228

*** ***

208.943 30.627

*** ***

29.261 13.731

*** ***

3.2. Polyps activity In the temperature range between 14 °C and 18 °C, all the species had more than 80% of the polyps open. At 20 °C, the percentage of open polyps for P. clavata and E. singularis decreased to approximately 60%, while for C. rubrum and E. cavolinii still 70% of the polyps was open. At 22 °C the percentage open polyps for all species was approximately 40% and at 25 °C approximately 15%. After three days at 25 °C the percentage of open polyps dropped to around 5% (Fig. 3). The same percentages of open polyps were observed during the respiration measurements, showing that the incubations did not affect the polyp activity patterns. Polyp activity and oxygen consumption showed a weak but significant positive correlation (Pearson's correlation, R2 = 0.221; P = 0.0016). Addition of food did significantly increase the percentage of open polyps in the temperature range between 14 °C and 20 °C (Table 3, Fig. 3). This result underlines the healthy condition of the experimental fragments. As long as polyps react to food supply, we can assume that a gorgonian is still alive. Feeding did not affect polyp activity at temperatures above 20 °C. 3.3. Necrosis pattern On the temperature range between 14 °C and 22 °C, the species does not show any sign of necrosis. After the first day under the highest temperature (25 °C), 80% of C. rubrum and 60% of P. clavata samples showed the first signs of necrosis: the colour of the middle parts of all fragments of P. clavata started to change from purple to greyish. After three days at 25 °C, two fragments of P. clavata completely lost approximately 30% of their coenenchyme (Fig. 4A). With regard to C. rubrum, three fragments showed the first signs of necrosis (change of colour) already on the first day at 25 °C and lost 10% of the coenenchyme after three days at 25 °C (Fig. 4B). E. cavolinii shows after three days at 25 °C only a reduction of colour from yellow to whitish while E. singularis did not show any necrosis. Table 2 Respiration rate (mg O2 g AFDM− 1 h− 1) (average ± SD) on different temperature regimes (between 14 °C and 22 °C) and under the highest temperature 25 °C maintained for one and for three days. Temperature

C. rubrum

P. clavata

E. cavolinii

E. singularis

14 °C 16 °C 18 °C 20 °C 22 °C 25 °C (1 day) 25 °C (3 day)

0.31 ± 0.14 0.34 ± 0.13 0.78 ± 0.24 0.92 ± 0.12 0.84 ± 0.12 0.2 ± 0.14 0.08 ± 0.01

0.41 ± 0.17 0.61 ± 0.10 0.91 ± 0.22 0.54 ± 0.09 0.53 ± 0.16 0.26 ± 0.10 0.09 ± 0.07

0.21 ± 0.08 0.51 ± 0.17 0.41 ± 0.13 0.61 ± 0.16 0.42 ± 0.19 0.28 ± 0.11 0.12 ± 0.08

0.10 ± 0.13 0.36 ± 0.33 0.66 ± 0.09 0.42 ± 0.15 0.16 ± 0.34 0.21 ± 0.09 0.23 ± 0.11

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4. Discussion Gorgonians are characteristic components of Mediterranean coralligenous community and have been reported as vulnerable to climate anomalies. Nevertheless, their physiological responses to temperature changes still remain poorly studied. Here, we analyzed the respiration rates and the corresponding Q10 values of four Mediterranean gorgonians under different temperature conditions, hereby also monitoring the polyp activity and the occurrence of necrosis. Respiration rates of all four gorgonian species tested here showed a similar bell-shaped trend when plotted against temperature. Respiration and polyp activity increased up to temperatures of 18 °C to 20 °C. At higher temperatures, polyps reduce their activity and the oxygen consumption decreases. This pattern mirrors the general model of temperature tolerance as adapted from Shelford's law of tolerance by Frederich and Pörtner (2000). The maximal respiration rate of P. clavata obtained in this study (0.91 ± 0.22 mg O2 g AFDM− 1 h− 1) is comparable with the value obtained in the field by Coma et al. (2002) (about 0.90 mg O2 g AFDM − 1 h− 1). For E. singularis, we measured an average respiration of (0.34 ± 0.22 mg O2 g AFDM− 1 h− 1) within the temperature range between 14 °C and 22 °C. This value is in very good agreement with the average respiration found by Brafield et al. (1965) for this species within the same temperature range in the field (0.33 mg O2 g AFDM− 1 h− 1). This compatibility of data indicates that the aquarium conditions used here appropriately mimicked natural conditions. The apparent optimal temperature for metabolism of P. clavata (18 °C) coincides with that described in the field about polyp activity (Coma et al., 2002) and with that of E. singularis. The polyp activity of E. cavolinii and C. rubrum started to decrease at 20 °C, indicating that 20 °C is the temperature at which the metabolism of these species becomes inhibited, although the small oscillations in oxygen consumption patterns of E. cavolinii noticed during the experiment did not allow accurate determination of the temperature at which respiration is maximal for this species. In the Mediterranean Sea, the summer season is considered potentially stressful to the benthic community (Coma and Ribes, 2003; Coma et al., 2009). In the summer season, temperature is maximal, whereas seston concentration and food availability are minimal (Gremare et al., 1997; Rossi et al., 2003; 2006). Under these conditions of reduced food availability and high temperature, many organisms cross a sort of aestivation point, reducing the activity levels and in some cases consuming the metabolic storages accumulated in winter–spring time. In Mediterranean sponges, such a summer dormancy is generally less evident. These organisms show an increase in respiration rate (Zocchi et al., 2001) and metabolic activity (Basile et al., 2009) in relation to temperature increase. Moreover, the growth dynamics of Mediterranean sponges throughout the year do not appear to be related to the availability of planktonic food (Coma et al., 2002). In contrast, many anthozoans show evident behavioural and metabolic responses to summer season. For example, from August to October, P. clavata consumes proteins, lipids and carbohydrates stored during spring (Rossi et al., 2006). In this period, P. clavata also reduces the activity of its polyps thus decreasing its food intake and hence, its oxygen consumption (Coma et al., 2002). This pattern occurs also in E. singularis, despite its symbiosis with zooxanthellae, which may affect lipid storage and consumption during summer season (Gori et al., 2007). The respiration rate of octocorals is largely determined by the rate of diffusion of oxygen through ectoderm and endoderm and by polyp activity (Fabricius et al., 1995; Coma et al., 2002; Coma and Ribes, 2003). The diffusion rate of oxygen is strongly influenced by the thickness of the diffusive boundary layer (DBL), a thin, stagnant water layer between the gorgonian tissue and the ambient water (Patterson and Sebens, 1989; Nakamura and van Woesik, 2001). The thickness of

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Fig. 2. Temporal patterns in oxygen consumption (mg O2 g AFDM− 1 h− 1) of the four species under different temperature regimes (between 14 °C and 25 °C at the first and third day) during the 8 h of incubation (480 min). (A) = Corallium rubrum; (B) = Paramuricea clavata; (C) = Eunicella cavolinii; (D) = Eunicella singularis.

the DBL is affected by water movement, hence, this factor must be considered both in laboratory and field experiments. Polyp activity affects respiration in two ways: polyp expansion maximizes the

diffusion and consumption of oxygen in the tissue, whereas the contraction of polyps reduces feeding efficiency and respiration (Sebens and De Riemer, 1977; Shick, 1990; Shimeta and Jumars,

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Fig. 3. Number of open and semi-open polyps (average ± SD) for each species under different temperature regimes (between 14 °C and 25 °C) before feeding (A) and after feeding (B).

1991; Fabricius et al., 1995). The results of our study are in line with this mechanism: temperature started to affect the polyp activity when it reached values close to the upper thermal tolerance limit. Polyps became inactive and the oxygen consumption decreased. The different polyp rhythms observed throughout the experiments remain unknown whether the expansion/contraction rhythms of polyps is related to endogenous or exogenous stimuli. Time-lapse photo analyses of E. cavolinii performed in the field, suggest the polyp

cycle rhythms to be endogenous, probably depending on metabolism regulation (Pronzato et al., 1994). Endogenous control of polyp activity has also been postulated for C. rubrum maintained in aquaria, which showed contraction/expansion cycles of approximately 300 s, (Russo et al., 1993; Santarelli et al., 1997). In the field, P. clavata shows rhythms that are likely related to exogenous stimuli, such as food concentration (Coma et al., 2004). The Eunicella species showed more regular rhythms, with a maximum always at the second hour. This

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Table 3 Analysis of variance of the open polyp percentage for the four species, on different temperature (between 14 °C and 25 °C) and feeding regimes (before and after feeding). (SS = Sum of Squares;*** = 0.001 b p; ** = 0.001 b p ≤ 0.01). Source

SS

df

Mean square

F

Sig.

Species Error (Species) Species ⁎ Temperature Error (Temperature) Species ⁎ Food Error (Food) Species ⁎ Temperature ⁎ Food

0.005 0.022 0.611 1.410 0.034 0.065 0.368

3 20 18 120 3 20 18

0.002 0.001 0.034 0.012 0.011 0.003 0.020

1.516

0.241

2.887

***

3.458

**

1.370

0.159

behaviour could be related to their lower metabolism lower in comparison with C. rubrum and P. clavata. In E. singularis, the presence of zooxanthellae seems to reduce the thermal stress, thus allowing a more constant respiration (Merle et al., 2009). Links between mass mortality events and global warming are well documented, both for coral reef ecosystems (Hughes et al., 2003) and for Mediterranean benthic assemblages (Cerrano et al., 2000; 2005; Cerrano and Bavestrello, 2008; Garrabou et al., 2009; Vezzulli et al., in press). According to several authors (Cerrano et al., 2005; Linares et

al., 2008), the mass mortality events primarily affected principally the density, growth and reproduction of Mediterranean gorgonians, thus considerably changing the specific composition and structure of coralligenous communities (Coma et al., 2006; Cerrano and Bavestrello, 2008; Scinto et al., 2009). Our study experimentally confirmed the relation between temperature and mortality. The four species studied showed slightly different thermal tolerance patterns. C. rubrum maintained high values of respiration until 22 °C, notwithstanding the fact that polyps started to close at 20 °C, while at 25 °C the oxygen consumption suddenly decreased and necrosis started. Exposure to 25 °C seemed to be critical for this species, which is in agreements to the results of Torrents et al. (2008). P. clavata proved to be the most sensitive to high temperatures, showing a peak of polyp activity at 18 °C and nearly complete retraction at 25 °C. This thermal sensibility is in agreement with the fact that this species has been severely affected by recent mass mortality events that were related to thermal anomalies (Cerrano et al., 2000; Cupido et al., 2007). P. clavata was also found to be very sensitive to virulent thermo-dependent bacteria, which further explains its vulnerability to high temperature (Bally and Garrabou, 2007).

Fig. 4. Sequences of imagine of C. rubrum and P. clavata under different temperature regimes: A) C. rubrum at 14 °C; B) C. rubrum after one day at 25 °C; C) particular of necrosis tissue and parasitic organism of C. rubrum after three days at 25 °C; D) P. clavata at 14 °C; E) P. clavata after one day at 25 °C; F) particular of the necrosis tissue and lost coenenchyme of P. clavata after seven days at 25 °C.

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E. cavolinii did not show signs of necrosis, even after being exposed for three days to 25 °C. Only an alteration of the coenenchyme colour was observed. This is in agreement with observations by Weinberg and Weinberg (1979) who reported an upper temperature limit of 27 °C for this species. According to our data, E. singularis appears to be the most tolerant to high temperatures. The upper distribution limit of E. singularis at 5 m depth further suggests a higher thermo-tolerance in comparison to the other species, like P. clavata, which has an optimum distribution between 35 and 80 m depth. The higher resistance of E. singularis is also confirmed by laboratory studies performed by Ferrier-Pagès et al. (2009). Moreover, Fava et al. (2010) evidenced also a higher resilience of this species by comparative experiments on several gorgonians species in the field. Anyway, according to Ferrier-Pagès et al. (2009), shallow populations are exposed to higher levels of irradiance than deep populations, weakening the organisms by producing a bigger oxidative stress due to higher production of free radicals. Also food availability need to be considered, particularly during the summer period, when less food is available (Coma et al., 2009) and when light and temperature levels are high (Gori et al., 2007). The presence of zooxanthellae may help to cope this poor season, providing an additional source of energy to E. singularis, thus reducing the demand for organic food. The proposed mediating role of zooxanthellae with regard to the thermo-tolerance of E. singularis is supported by our observations on necrosis phenomena, which started at 25 °C in C. rubrum and P. clavata but not in E. singularis. After 3 days at high temperature (25 °C), P. clavata and C. rubrum showed a sudden high percentage of necrotic tissue. E. cavolinii only exhibited an alteration of the coenenchyme colour (the first sign of partial mortality as reported by Coma et al., 2009), while E. singularis did not show any mark of necrosis and a low percentage of open polyps. These species-specific different responses to the most extreme temperature regime applied could be linked to the different abilities of the species to repair the injured coenenchyme. For example, E. cavolinii and E. singularis more rapidly regenerate their coenenchyme than P. clavata (Bavestrello and Boero, 1986; Cerrano et al., 2005; Fava et al., 2010). The Q10 value is considered as one of the best parameters to analyze the temperature dependence of a process (Brockington and Clarke, 2001; Gaudy and Thibault-Botha, 2007). The values of Q10 support the interpretation of our results, confirming that Mediterranean gorgonians have different metabolic performances at different temperature ranges: P. clavata and E. singularis showed increasing metabolic activity between 14 °C and 22 °C, E. cavolinii between 14 °C and 20 °C, and C. rubrum between 16 °C and 20 °C. In conclusion: 1) Oxygen requirements of gorgonians are an important aspect to consider when looking for causes of mass mortalities 2) All species studied exhibited a similar bell-shaped trend with a temperature limit (between 18 °C and 20 °C) above which polyps reduced their activity and the oxygen consumption decreased; 3) The metabolism of the four species studied decreases substantially with high temperature, above a definite threshold; 4) The relation between temperature and mass mortality was experimentally confirmed: an exposure of 25 °C for three days seems to be critical for C. rubrum and P. clavata that showed a sudden high percentage of necrotic tissue and for E. cavolinii that exhibited an alteration of the coenenchyme colour. No signs of necrosis were observed in E. singularis at that temperature, confirming that this species has a higher upper temperature limit. Acknowledgements This project has been supported by the WIAS scholarship (Aquaculture and Fisheries Group, Wageningen University) and the European Union (project CORALZOO 012547). The authors thank Marzia Sidri,

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