Erschienen in: Marine Environmental Research ; 140 (2018). - S. 444-454 https://dx.doi.org/10.1016/j.marenvres.2018.07.006
Transcriptional response of the heat shock gene hsp70 aligns with differences in stress susceptibility of shallow-water corals from the Mediterranean Sea
1 1 Silvia Franzellittia,•, , Valentina Airib• , Diana Calbuccia, Erik Carosellib, Fiorella Pradab, 3 Christian R. Voolstrac, Tali Massd, Giuseppe Falinie, Elena Fabbri , Stefano Goffredob
•Anima/ and Environmental Physiology Laboratory, Depanment of Biologica~ Geological and Environmentnl Sciences, University of Bologna, via S. Alberto 163, 1-48123, Ravenna, ltaly b Marine Science Group, Depanmenc of Biologica~ Geological and Environmentnl Sciences, Universily of Bologna, Via F. Selmi 3, 1-40126, Bologna, ltaly c Red Sea Research Center, Division of Biological and Environmerual Scitnct and Engineering (BESE), King Abdullah University of Science and Technology (KAUST), 1'huwal, 23955·6900, Saudi Arabia d Depanntent of Marine Biology, The Leon H. Chamey School of Marine Sciences, University of Haifa, Multi Purpose Boulevard, ML Camiet Haifa, 3498838, Israel • Depanment of Chemistry "Giacomo Ciamician", University of Bologna, via F. Selmi 2, 1-40126, Bologna, ltaly
ARTICLE INPO ABSTRACT
Keywords: Shallow-water corals of the Mediterranean Sea are facing a dramatic increase in water temperature due to Thermal stress climate change, predicted to increase the freq uency of bleaching and mass mortality events. However, suppo Coral sedly not all corals are affected equally, as Lhey show differences in s1ress susceptibility, as suggested by phy· Gene expression siological outputs of corals along temperature gradients and under controlled conditions in terrns of reproduc Heat shock protein tion, demography, growth, calcification, and photosynthetic efficiency. In this study, gene expression and PhysiologicaJ plasticity Climate change induction of a 70-kDa heat shock protein (HSP70) was analyzed in five cornmon shallow-water hard corals in the Mediterranean Sea, nan1ely Ascroides calycularis, Balmtophyllia europaea, CaryophyUia inomata, Cladocora cae spitosa, and leptopsanunia provoli The main ain1 was to assess the contribution of this evolutionary conserved cytoprotective mechanism to the physiological plasticity of these species that possess different growth modes (solitary vs colonial) and trophic strategies (zooxanthellate vs azooxanthellate). Using quantitative real-ti me PCR, in siru hsp70 baseline levels and expression profiles after a heat-shock exposure were assessed. Levels of hsp70 and heat stress in.duction were higher in zooxanthellate than in azooxanthellate species, and different heat stress transcriptional profiles were observed between colonial and solitary zooxanthellate corals. On the whole, the hsp70 transcriptional response to heat stress aligns with stress susceptibility of the species and suggesrs a contribution of trophic stra1egy and morphology in shaping coral resilience ro stress. Understanding these molecular processes may contribute to assess the potential effects and relative resilience of Mediterranean corals under climate change.
1. Introduction tropics and in the Mediterra nean Sea (Altieri et al., 2017; Jimenez et al., 20ll6; Rodolfo-Metalpa et al„ 2006b). The Mediterranean Sea is Marine ecosystems are declining worldwide due to global climate warming a t two to three times the rate for the global ocean (Vargas· change and other anthropogenic impacts (Hughes et al., 2018). These Yanez et al„ 2008), showing an in creased occurrence of hot extremes by rapid changes alter the structure of habitats and communities and cause 200- 500% throughout the region (Diffenbaugh et al., 2007). This is due the loss of many species. Of particular concern is the decline of scler to i ts position in the transition between the arid clim ate of North Africa actinian corals triggered by thermal stress (Hoegh-Guldberg et al„ and the temperate climate of central Europe, which renders Medi· 2007). Rising sea surface temperature is linked to coral bleaching and terranean climate vulnerable to even relatively moderate modifications mass mortality events in both symbiotic and non-symbiotic corals in the of the general circulation (Giorgi and Lionello, 2008). Furthermore, its
• Corresponding author. E-mail address: [email protected] (S. Franzellitti). 1 These authors equally contributed to the work performed.
Konstanzer Online-Publikations-System (KOPS) URL: http://nbn-resolving.de/urn:nbn:de:bsz:352-2-12jnkvq0v16cx6 semi-enclosed nature and the restricted water exchanges with the 2008; Morris et al., 2013), and amongst the primary early responders to Atlantic Ocean results in a hydrological residence time of about 100 stress in corals (Louis et al., 2017; Traylor-Knowles et al., 2017). As a years and a great capacity to store heat (Diffenbaugh et al., 2007). In consequence, many studies assessed hsp70 expression in tropical corals deed, climatic models predict a rapid average warming in the Medi under different abiotic and biotic cues, including temperature (Maor terranean region (about 2.5 'C over the 21" century under the RCP8.5 Landaw et al., 2014; Seveso et al., 2014; Zhang et al., 2018), pH/pC02 projection; Shaltout and Omstedt (2014)) along with a greater occur (Moya et al., 2015), salinity (Ellison et al., 2017), pollutants (Jovanovic rence of extreme temperature events (Giorgi and Lionello, 2008). Under and Guzmän, 2014; Overmans et al., 2018; Venn et al., 2009), bacterial such a scenario, not all corals are thought to be affected equally: during infections (Brown et al., 2013; Seveso et al., 2016), or host-symbiont mass coral bleaching events, survival of scattered colonies suggests that interplay to maintain holobiont homeostasis (Levy et al., 2011; Rosic some groups of corals may possess inherent physiological tolerance to et al., 2014b). These studies suggest that cnidarian hsp70 gene ex environmental stress (Marshall and Baird, 2000; Oliver and Palumbi, pression and function is similar across multiple stressors and compar 2011; West and Salm, 2003). Therefore, questions remain about the able to vertebrates (Kvitt et al., 2016). Furthermore, hsp70 up-regula underlying mechanisms leading to differences in thermal tolerance, and tion at the initial stage of the stress response appears to be a common about the relative contributions of genotypic adaptation (where selec protective mechanism to avoid the onset of severe pathological condi tion drives differences in susceptibility over evolutionary times scales), tions or to postpone bleaching (Maor-Landaw et al., 2014; Rosic et al., and phenotypic acclimatization ( where organisms respond to extremes 2014a). using their existing genomic repertoire) (Palumbi et al., 2014). Studies on hsp70 expression in relation to environmental variables Changing environmental conditions may be physiologically toler or to symbiosis in Mediterranean corals are lacking, and hsp70 se able, allowing acclimatization or adaptation, or may be intolerable, quences are not available for the species investigated in this study. An promoting change in phenology (timing of annual events), or massive exception is represented by B. europaea, for which a recent investigation bleaching, death and local extinction (Parmesan, 2006). Physiological based on whole transcriptome sequencing reported hsp70 transcrip performances and responses are the principal determinant of a species tional response to temperature and pH combined exposures (Maor tolerance to environmental variability and, as such, they may change Landaw et al., 2017). The comparison with the tropical Stylophora pis the ability of individual corals to cope with changing conditions tillata suggested that B. europaea is endowed with more efficient pro (Somero, 2012). Physiological plasticity describes the process of tuning tective and metabolic processes to cope with the high thermal varia phenotypic traits of organisrns within their lifetime allowing them to bility of Mediterranean environments (Maor-Landaw et al., 2017). To live in varying environments (Gates and Edmunds, 1999). In this re our knowledge, this is the first study reporting a comparative multi gard, investigations of the regulatory mechanisms governing stress re species analysis of the HSP70 response to thermal stress in temperate sponses by monitoring changes of mRNA expression profiles may pro corals, which attempts to set the basis for future investigations on the vide early-warning molecular markers of physiological plasticity and potential regulatory mechanisms behind physiological resilience and represent an important source of information on the mechanisms un accJimatization capabili ties in corals from the Mediterranean Sea. We derlying the differences in coral physiological resilience (Dixon et al., investigated transcriptional regulation of a stress inducible hsp70 by 2015; Poli et al., 2017; Seneca and Palumbi, 2015). concomitant analysis of expression levels under field conditions and Although studies on tropical corals disclosed some molecular me following short-term heat stress experirnents, to infer possible influ chanisms underlying acclimatization and coordinated modulation of ences of peculiar physiological features and diverse life history traits. gene expression as a reaction to envi ronmental stress (Bay and Palumbi, Gai ning this knowledge is a first important step towards understanding 2017; Bellantuono et al„ 2012; Gutner-Hoch et al., 2017; Maor-Landaw the processes involved in species resilience to heat-stress events and et al., 2014), similar information on tem perate corals of the Medi may disclose the relevant threats involved in their decline. terranean Sea are largely lacking. To fill this gap, the present study assessed expression of a hsp70 gene product under baseline and heat 2. Methods stress conditions in five Mediterranean shallow-water coral species re presenting different growth modes (colonial: Astroides calycularis and 2.1. Coral sampling and experimental design Cladocora caespitosa; solitary: Balanophyllia europaea, Caryophyllia in omata, and Leptopsammia pruvoti) and trophic strategies (zoox During May 2016, 60 nubbins (from 6 colonies) of C. caespitosa and anthellate: B. europaea and C. caespitosa; azooxanthellate: A. calycularis, 60 polyps of B. europaea, C. inomata, and L. pruvoti were collected by C. inomata, and L. pruvoti) (Fig. 1). An overview of population dynamics SCUBA diving at Calafuria (Leghorn; 43°28.4'N, 10' 20'E) (Fig. 2). and physiological traits (i.e., reproduction, linear extension rate, net Nubbins (60 from 6 colonies) of A. calycularis were collected at Ponza calcification, and photosynthetic efficiency) of these species in relation Island (40'54'N l2°58'E) (Fig. 2). Samples were collected from sites to increasing temperature is provided in Fig. 1, as a means to infer their characterized by high population density at 5-30 m depth, and all relative tolerances to stress. For example, azooxanthellate species C. samplings were performed at the same time of the day (early morning), inomata and L. pruvoti seem qui te tolerant to temperature increases to minimize possible biases due to Hsp70 die! modulation (Levy et al., compared to their sympatric zooxanthellate species B. europaea and C. 2011 ; Sevcso et al., 2018). Upon collection, 12 randomly selected single caespitosa, while A. calycularis responds negatively to increasing tem polyps or fragments of colonies (2 nubbins per colony) from each spe peratures (Fig. 1). cies were immediately immersed in a suitable volume of the RNA later The 70-kDa heat shock proteins (HSP70s) are molecular chaperones stabilization reagent (Sigma-Aldrich, Milan, Italy) and frozen at - 80 'C and havc vital cytoprotective functions (Feder and Hofmann, 1999). for furthcr analysis of transcriptional lcvels of coral hsp70 under in situ They are involved in protein folding, unfolding, sorting transport, and baseline conditions. The remaining samples were immediately put in assembly of complexes. They also protect cells from apoptosis and refrigerated seawater and transported to the aquarium system of the proteotoxic stressors (Hartl et al., 201 1). During exposure to elevated Departrnent of Biological, Geological and Environmental Sciences temperatures, protein misfolding, aggregation or disruption of regula (BiGeA) of the University of Bologna. They were acclimatize for 40 days tion and disassembly of multiprotein complexes may occur, leading to at a constant water temperature of 17 ' C ± 0.5 'C matching the subsequent activation of signaling pathways triggering a stress-related average temperature at the collection sites (Fig. 28 and C). Zoox HSP70 induction (Richter et al., 2010). Through their cytoprotective antheUate species (8. europaea and C. caespitosa) were maintained at a functions, HSP70 are thought to restore proteolytic homeostasis. photoperiod of 15 h:9 h light:dark daily cycles to match the natural HSP70s are broadly studied, highly conserved molecular mediators photoperiod at the collection sites. Azooxanthellate species (A. calycu of environmental acclimatization in marine organisms (Fabbri et al., laris, C. inomata, and L. pruvoti) were maintained under dimmed light
445 Fi.g. 1. Mediterranean shallow water corals used in this study alongside phylogenetic relation ships and physiological outputs in relation to temperature. Data on population density, reproductive ef ficiency, linear extension rate, and net calcification refer to field studies conducted along temperature gra dients, and photosynthetic efficiency refers to experiments under con troUed conditions. Shapes indicate the species temperature-related rc sponse (square: no change; triangle: Cladocora Caryophyllia Balanophyllia Astroides Leptopsammia decrease), colors indicate the direc caespitosa inornata europaea ca/ycularis pruvoti tion of temperature change reported in the referred papers, fro m lower (blue) to higher (red) temperarures. Growth mode Colonial Solitary Solitary Colonial Solitary Zoox: zooxanthellate. Azoox: azoox anthellate. • no data available. Coral picrures are fro m Franceseo Sesso Trophic strategy Zoox Azoox Zoox Azoox Azoox (Caroselli et al., 2015a; Caroselli et al„ 2015b; Caroselli et al„ 2012b; Goffredo et al„ 2009; Goffredo et al„ 2008) (For interpretation of the re Population density ferences to color in this figure legend, the reader is referred to the Web ~ version of this article).
Reproductive efficiency ~ Linear extension rate ~ Net calcification rate ~ ~ Photosynthetic efficiency• ~ References Ro • Noehange ~ Decrease conditions to mimic their natural environment. During the acclimati sche me and experimental parameters were selected accord ing to our zation period, corals were fed twice a week with a commercial pla nkton past experience on marine invertebrate investigations on the HSP70 preparation (Elos Coral Foods SvC). response (Franzellitti and Fabbri, 2005; Piano et al., 2005, 2004). Thermal stress experiments were performed using a short-term heat Briefly, this experimental setup was chosen to analyze the different shock treatment at a relatively high temperature (here 32 ·c. about 5 ·c capabilities of the selected species to promptly increase hsp70 tran above average ambient values) followed by 0-24 h of post-stress re scription within short-term in tense heat stress exposures. lt also ac covery at the coral acclimatization temperature (Fig. 3). This exposure counts for the specific regulatory features of stress-inducible Hsp70s, 446 25 ~ 3 -0 ~ 20 c~ iil 42° 0 15- Tyrrhenian Sea 30 38° Fig. 2. Map of the sampling sites at Calafuria and Ponza Island (Thyrrenian Sea, Italy}. (A) The map of the sampling locations was generated using the OpenStreetMap database (http:// www.openstreetmap.org/ copyright). Mean satellite-derived daily sea surface temperature (SST) profiles at (B) Calafuria and (C) Ponza sampling locations (across a 2-year period spanning the sampling time point) were retrieved using the Copernicus Marine Service Product (http://marine. copemicus.eu/ }, and visuaJJzed through Panoply ver 4.8 (http://www.giss.nasa.gov/ tools/ panoply/ }. Sampling period is showed (grey area}. Average SSTs within the sampling period were (mean :t SO) 16.9 :t 0.5 ·c (Calafuria) and 17.4 :t o.rc (Ponza) (For interpretation of the references color in this figure legend, the reader is referred to the Web version of this article). within the earlier phases of the stress response, after which further sampled, randomly pooled into 3 samples (2 nubbins/polyps for each compensative and/or adaptive processes may be activated (Franzellitti sample}, immediately immersed in RNA later solution (Sigma Aldrich, et al., 201 O; Franzellitti and Fabbri, 2005; Maor-Landaw et al., 2014; Milan, Italy), and stored at - 80 ' C fo r subsequent procedures (see Morris et al., 2013; Rosic et al., 2014a). below). Thermal stress experiments were settled to allow 0 h and 24 h After acclimatization, 12 nubbins/polyps for each species were post-stress recovery samplings at the same time at which the field collected to account for differences in animal physiological status at the sarnplings were performed. onset of the experimental exposure to thermal stress, thus representing the control condition of the experiment (2 nubbins/polyps were pooled 2.2. RNA extraction and cDNA preparation together to give 6 samples per species; N = 6). The remaining samples were divided in different transparent glass beakers, to facilitate their Nubbin/polyp pools were homogenized in a suitable volume of the manipulation during the experiments, and transferred to an experi Tru Reagent (Sigma Aldrich, Milan, Italy) according to Barshis et al. menta l tank where the water temperature was raised to 32 •c. The (2013). Total RNA was further extracted using the OirectZol kit (Zymo samples were maintained at 32 •c for 2 h, and then all owed to recover Research, Freiburg, Germany) according to the manufacturers' protocol. fo r O·, 2-, or 24-h post-stress periods at the accl imatization temperature DNAase 1 treatrnent was performed within the RNA extraction proce (17 ' C) (Fig. 3). At each time-point 6 nubbins/polyp per species were dures according to the manufacturers' instructions (Zymo Research, Temperature (' Cl Fig. 3. Scheme of the experimental design for the 17' C 32' C 17' C coral heat shock exposure. To: time zero of the heat stress exposure, before thc onset of thc tcmperature challenge in the hear Stress treatment. Sampling time Exposure points refer to the recovery periods (R) post-stress (For stages ~cNmotim~?2------Post---..--....,- Control T0 Oh R 2h R 24hR Sampling time-points 447 Freiburg, Germany). RNA concentration and quality were confirmed Poli et al. (2017). The obtained nucleotide sequences were then aligned using the Qubit system with the Qubit RNA assay kit (Thermo 5cien and analyzed against this dataset using the MEGA 6 program package tific, Milan, Italy) and electrophoresis using a 1.2% agarose gel under (Tamura et al., 2013) to test the degree of sequence identity with other denaturing conditions. RNA integrity was evaluated based on the pre coral hsc70 and hsp70 genes. sence of clear 285 and 185 ribosomal RNA bands in the electrophoresis, and by confirming that 28S band intensity was at least double that of 18S. A further qualitative analysis was performed by measuring the UV 2.4. qPCR assays absorbance spectra of the samples (A. = 200-340 nm). Absorbances (A) Levels of hsp70 transcripts was assayed by quantitative Real Time at 260, 230, and 280 nm were used to calculate the ratio A2ro/A 280 addressing the occurrence of protein contaminations (cut-off va Polyme.rase Chain Reaction (qPCR) assays and using a protocol for the absolute quantification of the target transcripts. This approach was lues > 1.8 and < 2.0), and the ratio A260/A230 addressing the occur rence of contaminants that may be present in the samples, such as chosen to overcome the challenges in identifying stably expressed re guanidine thiocyanate, which is a component of the TRI Reagent (cut ference transcripts, which is a demanding issue in relative quantifica off value > 1.7). tion studies (Bustin et al., 2013). Reliable reference transcripts are re First strand cDNA for each sample was synthesized from 600 ng ported only for model tropical corals including Acropora and Pocillopora total RNA using the iScript supermix following the manufacturer's species (Oshiro et al., 2013; Overmans et al., 2018; Pagarigan and protocol (Bio-Rad Laboratories, Milan, ltaly). Takabayashi, 2008; Rosic et al., 2014a; van de Water et al., 2015). There is poor or no genomic/ transcriptomic data for the species con sidered in this study, making it di fficult to select and statistically vali 2.3. Cloning and sequencing date any choice of best-performing reference transcripts (Balbi et al., 20ll6; Franzellitti et al., 2015; Rosic et al., 2014a). The cDNAs corresponding to the coral hsp70 mRNAs were amplified A universal hsp70 primer pair was designed with the Prinler Express by a semi-nested PCR protocol using degenerated primers designed on sofltWare (Life Technologies, Milan, Jtaly) using nucleotide sequences conserved regions of a multiple alignment of homologous coral protein obtained in this study (Table 1) and exhibiting low sequence homology sequences retrieved from the GenBank and the Reefgenomics.org to hsp70 from Symbiodinium clade A and C to avoid non-target ampli (Bhattacharya et al., 2016; Liew et al., 2016) databases (Table 1). fication. qPCR standards for the target transcript were prepared by The multiple alignment was done using the MEGA 6 software (Tan1ura serial dilution of the linearized plasmid DNAs containing the specific et al., 2013) and a dataset of coral Hsp70 protein sequences reported by transcripts to obtain a standard curve of Cr values vs the logarithmic Poli et al. (2017). For each coral species, cDNA employed at this stage DNA amount. R2 va lues for all standard curves were > 0.99. Absolute were synthetized from pooled heat-stressed coral total RNA samples mRNA abundance was calculated from the Standard curves and plotted (2 h at 32 ·c + 2 h of post-stress recovery). In the first PCR run, the as copy number ng- 1 RNA (mean :t SEM). Reactions were performed primer pair CoHSP70Fl (5'-ATHGAYCTNGGNACNACNTA-3') and in a final volume of 10 µL containing 5 µL iTaq Universal SYBR Green CoHSP70R2 (5'-AGNACNSWNACRTCRAANGT-3') was used. In the Supermix with ROX (Bio-Rad Laboratories, Milan, Italy), 2 µL diluted second PCR run, products of the first PCR run were used as templates cDNA or plasmid DNA, and 0.2 µM specific primers. A control lacking for the reaction with the CoHSP70Fl forward primer and the (intemal) the DNA template (no-template) and a minus-reverse transcriptase (no reverse primer CoHSP70Rl (5'-NCCNGCRTCYTTNGTNGCYTG-3'). In RT) control were included in the qPCR analysis to ensure the specificity both runs, PCR reactions were performed in a 25 µL volume containing of the amplification. 3 µL of cDNA template or 1 µL of PCR product, 0.5 µM each primer, Technical replicates were performed both within each run/ plate 1.5 mM MgCb, 0.2 mM each dNTP, and 0.5 units of RedTaq DNA (samples in duplicate, qPCR standards in triplicate) and between dif polymerase (Sigma Aldrich, Milan, ltaly). The thermal profile consisted ferent runs/ plates (Standards and samples replicated on different of an initial denaturation step (95 •c, 1 O min), followed by 38 cycles of plates). Equal loadings within each qPCR reaction were ensured by denaturation (94 ' C, 20 s), annealing (52 •c, 45 s) and extension (72 •c, checking the amounts of each standard and cDNA sample using the 2 min), and a final extension step (72 •c, 10 min). PCR products were Qubit system with Qubit0 dsDNA H5 (H igh Sensitivity) assay kit separated on a 2.5% wide range agarose gel (Sigma Aldrich, Milan, (Thermo Scientific, Milan, Ita ly). ltaly) using TBE buffer (1 M Tris, 0.9 M boric acid, and 1 mM EDTA) Amplification was detected with a StepOne real time PCR system and stained with the GelRed staining solution (Biotium, VWR Interna (Llfe Technologies, Milan, ltaly) using a Standard "fast mode" thermal tional, Milan, Jtaly). Gels were analyzed with the Gel Doc'" EZ System protocol. For each target mRNA, melting curves, gel pictures, and se and the lmageLab software (Bio-Rad Laboratories, Milan, Jtaly). PCR quences of PCR products were analyzed to verify the specificity of the products of the expected size were cloned into a pSC-A plasmid vector amplified products. PCR products for each species were cloned and (Agilent, Milan, Italy) using the StrataClone PCR cloning kit (Agilent, sequenced. About 10 positive clones for each PCR product were se Milan, Italy). No PCR product was detected from negative controls quenced that confirmed that they were identical to the respective nu (reactions without reverse transcriptase added). The screening of clones cleotide sequences originally employed for designing the selected was performed th rough EcoRI digestion of plasmid DNA, after plasmid primer pairs (L. pruvoti sequences showed a mean 80% homology with extraction with the GenElute plasmid miniprep kit (Sigma Aldrich, sequences from other 4 selected species). Although potential off-target Milan, ltaly). Sequencing of both Strands of the plasmid DNA from amplification of Symbiodinium traru;cripts cannot be completely ruled positive clones was performed by 5anger sequencing (BMR Genomics, out, the low sequence homology to Symbiodnium hsp70 of the employed Padoa, Jtaly). A dataset of available coral hsp70 nucleotide sequences primer pair in combination with the melting curve analyses confirmed was built using the corresponding protein sequence dataset reported by that the employed qPCRs mostly account for changes in mRNA Table 1 Parameters of primers employed in the real-time qPCR assays. Prlrner name Sequence 5'-3' Ampllcon slze (bp) TmCC) Ampllßcatlon efficlency' (%) MC_HSP70F CTTTCcrTCCGAGATAGTcrTCAG 107 62 Samples: 109.8 :!: 10.9 MC_HSP70R TCCCGTACAGGTTGGTAAAG 62 Standards: 108.3 :!: 9.6 • Amplification efficiencies for samples and standards are reported as mean :!: SO. Tm: melting temperature. 448 B. europaea 2 0 post-stress recovery time Fig. 4. Expression of hsp70 in Mediterranean shallow-water corals. (A) In situ hsp70 baseline expression in B. europaea, A. calycularis, C. inomata, C. caespitosa, and L. pruvoti. Box-and-whisker plots represen! median, upper and lower quartiles (N = 6) of h.VJ70 copy number normalized over nanograms of total RNA employed in each PCR reaction. Different letters indicate statistical differences (P < 0.05, Mann-Whytney U test). (8-F) Expression of hsp70 foUowing an acute heat shock treatment (2 h at 32 'C) and several post-s1ress recovery periods (0- 24 h) at 17 'C. Levels of hsp70 are expressed as the relative variation (fold change) to conrrols (means ± SEMs; N = 3). •p < 0.05 vs ctr (Mann-Whytney U tes1). Species in green = 8. europaea and C. caespitosa (zooxanthella1e); species in brown = A. calycularis, C. inomata, and L. pruvoli (azooxantheUa1e) (For Interpretation of the references to color in this figure legend, the reader is referred to the Web version of this articleJ. expression profiles of the coral species. Euclidean distance (Anderson et al., 2008). An ordination analysis The amplification efficiency was calculated using a dilution series of (carried out by Principal Coordi nates, PCO) and data clustering using samples cDNAs or of the linearized plasmid DNAs for the qPCR Stan the PRIMER v6 software was perfo rmed on data from in situ hsp70 dards (Table 1). PCR efficiencies of the standards were not significantly baseline levels in the selected coral species and pattern of hsp70 over different from those of the samples (P > 0.05, Mann-Whitney U test). expressions followi ng heat stress. Log-transformed hsp70 levels under in A standard curve was included in every PCR run. Consistency of tech situ baseline conditions and hsp70 induction across the post heat-stress nical replicates within each run was checked as a default operation by recovery period (expressed as the calculated AUC, Area Under the the StepOne software (Standard deviations between replicates < 0.5). Curve of the hsp70 transcriptional profiles for each species; Fig. Sl and Consistency of data between different runs was assured by analyzing Table Sl, Supplemental material) were employed to calculate similarity reproducibility of Cr values obtained from qPCR Standards or randomly matrices based on the Euclidean distance (999 permutations). selected samples from any experimental condition (P > 0.05, Mann Whitney U test). Consistency of parameters of standard curves be 3. Results longi ng to different runs was also assessed (P > 0.05, Mann-Whitney U test). 3.1. Sequencing of hsp70 cDNAs 2.5. Statistical analysis Using degenerated primers and a semi-nested PCR approach, se· quences for partial hsp70 cDNAs from B. europaea (15), A. calycularis Univariate comparisons of qPCR data from in situ baseline evalua (17'), C. inomata (11), and C. caespitose (9) were obtained. Positive tions and heat stress experiments were performed using the non-para clones included a partial ORF of about 450 bp corresponding to the mctric onc-way ANOVA (Kruskal-Wallis tcst) followcd by thc Mann ccntral fragmcnt of the hsp70 coding rcgion. Rcprcscntativcs of HSP70 Whitney U test (P < 0.05), after deviations from parametric ANOVA sequences have been submitted to the GenBank database (A. calycu assumptions were veri fied (Normality: Shapi ro-Wilk's test; equal va r laris: MH644814; B. europaea: MH618777; C. caespitosa: MH644815; C. iance: Bartlett's test). Data analyses were perfo rmed usi ng GraphPad inom ata: MH618776). The sequences were aligned together with a Prism 6 software (GraphPad Inc.). Permutation multivariate analysis of dataset of coral hsp70 nucleotide sequences retrieved from GenBank variance (PERMANOVA) using the PERMANOVA + add-on in PRIMER and the Reefgenomics.org databases [the complete dataset corresponds v6 (Anderson et al., 2008) was performed on in situ hsp70 baseline to that reported by Poli et al. (2017) for related protein sequences]. expressions. Log-transformed hsp70 copy number variations were used Sequences of hsp70 for the analyzed species shared a major sequence to calculate similarity matrices based on the Euclidean distance (999 identity (about 62%) with hsp70s from members of the Pori.tes fa mily, permutations). Factors considered were "growth mode" and "trophic while only an average 30% sequence identity with the available con strategy", that were used to calculate similarity matrices based on the stitutive Scleractinian hsc70s. Amongst the selected Mediterranean 449 Table 2 PERMANOVA results on basal hsp70 expression in the selectecl Mecliterranean corals under field conditions (998 perrnutations). ASTRO @ Source df Pseudo-F P(perm) Growlh mode 3.622 0.069 Trophic strategy 98.653 0.001 Bel /. Growlh mode x Trophic strategy 11.935 0.003 ...... 1 ' \ \ df. degree of freedom; Pseudo-F: F value by permutation (Anderson et al., 8 \ c ~ 2008); P (perm): probability of pseudo-F. 0 ~ ',_!J - 1 corals, B. europaea and A. calycularis showed the highest pairwise se -2 -1 0 2 quence identity score (about 71 %), while the lowest score (about 53%) PC01 (86.4% of total varlatlon) was between B. europaea and C. inomata. Fig. 5. Principal coordinates ordination (PCO) bi-plot of the hsp70 re sponse with super-imposed cluster analysis (grey dotted lines) by condi 3.2. Becween-species differences of in situ hsp70 baseline expression Levels tioo (i.e., different morphotype and different symbiotic partnership) (Euclidean Oistance resemblance matrix; 999 permutations). PCO and d uster analyses were performecl on a resemblance matrix generated from both Expression of hsp70 transcript was assessed in B. europaea, A. caly in si.ru hsp70 baseline expression and hsp70 induc1ion across different periods of cularis, C. inomata, C. caespitosa, and L. pruvoti using an absolute post heat-stress recovery (area under the curve, AUC, showing the hsp70 fold quantitative real-time PCR assay (Fig. 4A). Results showed that the change variations across the post-stress recovery period; Fig. SI and Table. SI, hsp70 transcripts were expressed in all samples of each species. The Supplemental material). Green circles indicate the zooxanthellate species, or zooxanthellate species B. europaea and C. caespitosa displayed sig ange circles indicate azooxanthellate species. ASTRO: A. calycularis; BEU: 8. nificantly higher in situ hsp70 levels compared to the azooxanthellae europaea; CARYO: C. inomata; Cl.AD: C. caespiU!Sa; LEPTO: L. provoti (For in· species. The only exception was represented by the azooxanthellate C. terpretal ion of the references to color in this figure legend, the reader is referred inomata, which showed in situ hsp70 baseline levels si milar to those of to the Web version of this article). B. europaea (Fig. 4A). PERMANOVA analyses of hsp70 expression re vealed a significant interaction of the factors "trophic strategy" and and no significant variations at 0 and 24 h post-stress recovery (Fig. 4F). "growth mode" (Table 2). Pairwise comparisons by permutation t-tests A PCO analysis was applied to determine whether the factors showed significantly higher in situ hsp70 baseline expression levels of "growth mode" and "trophic strategy" could discriminate samples by the colonial morphotype within zooxanthellate species (Fig. 4A), while means of variations of in siru hsp70 baseline expre.ssions as weil as of no significant differences within azooxanthellate corals were found species·specific differences of hsp70 response in the post heat-stress (Table 3). recovery experiment (Fig. 5). The latter parameter was measured by assessing the area under the curve describing hsp70 transcriptional 3.3. Transcriptional responses and inter-species differences of the hsp70 profiles across the recovery period post-stress (Fig. Sl and Tablc. SI, expression profiles under a heat shock treatment Supplemental material). Two principal coordinates (PCOl and PC02) were observed to explain 100% of total variance (86.4% and 13.6%, Changes of hsp70 expression levels were evaluated following an respectively). The super-imposed data clustering showed that sarnples acute heat shock (2 hat 32 °C) and different periods of post-stress re from zooxanthellate B. europaea and C. caespitosa formed one group covery (0 h, 2 h, 24 h) at the control temperature (l 7°C) (Fig. 3). For with marked separation from the azooxanthellate species (Fig. 4). each species, values were norrnalized against mRNA expression levels at Am.ongst the azooxanthellate corals, A. calycularis showed separation the onset of the experimental exposure to thermal stress (control sam from C. inomata and L. pruvoti, likely due to the remarkably different ples, ctr), achieved after a 40-days acclimation period to laboratory hsp70 transcriptional profile (i.e. higher AUC values) during the 24-h conditions (Fig. S2, Supplemental material). pos.t stress recovery (Fig. 4). Among zooxanthellate species, B. europaea showed significantly increased hsp70 expression at 2 h post-stress recovery (Fig. 48), with 4. Discussion levels decreasing thereafter, while C. caespitosa showed no significant variation of hsp70 levels (Fig. 4C). Amongst azooxanthellate species, A. In this study, we explored whether five shallow-water coral species calycularis showed the highest hsp70 fold-change increase, with levels of the Mediterranean Sea with di fferent growth modes (solitary vs co being significantly higher than controls from 0 h post-stress recovery lonial) and trophic strategies (zooxanthellate vs azooxanthellate) have and reaching a 3.5-fold and 3-fold up-regulation at 2 h and 24 h post differential transcriptional profiles of their hsp70 expression in situ and stress recovery, respectively (Fig. 40). Levels of hsp70 in C. inomata in response to thermal stress. Notably, these species showed distinct were significantly increased compared to controls at 0 h post-stress and susceptibilities towards ternperature (Fig. 1). recovered control va lues thereafter (Fig. 4E). L. pruvoti showed a small Oegenerated primers designed based on the multiple alignment of though signiflcant hsp70 over-expression at 2 h post-stress recovery, homologous hsp70 coral protein-coding nucleotide sequences allowed thc idcntification of partial hsp70 scqucnccs in B. europaea, /\. calycu Table 3 laris, C. inomata, C. caespitosa. Sequence alignments and identity scores Permutation t-tests through PERMANOVA pairwise comparisons fo r the effects showed that these partial sequences encoded heat inducible HSP70s of the factor "growth mode" on basa l hsp70 expression amongst zooxanthellate displaying a 60%-68% sequence identity with inducible HSP70s from or azooxanthellate species. Porices auscraliensis, while only about 30% sequence identity with the Palrwise comparisons (colonial vs P(penn) Unique permutaUons available constitutively expressed scleractinian hsc70s. These partial solltary polyp) seq·uences were ernployed to design specific primer pairs to be used in absolute qPCR protocols. Validation by electrophoresis, cloning, and AWoxanthelJate species 1.02 0.299 747 Zooxanthellate species 5.55 0.002 747 sequencing of qPCR products demonstrated that the same set of primers allowed hsp70 transcriptional analysis in all the five Mediterranean t: t-Statistics; P(perm): probability oft. coral species investigated in this study and detected no arnplification of 450 algal symbiont hsp70 transcripts. Consequently, these primers were relatively higher heat fluxes at the water-polyp interface compared to used to assess hsp70 expression across further Med iterranean coral the (bigger) colonial C. caespitosa, thus showing higher hsp70 induction. species. Cladocora caespitosa has been subjected to recurring mass bleaching Transcript levels of hsp70 were evaluated immediately after coral and mortality events due to anomalous heat waves in different areas of sampling to account for differences in mRNA expression under in sit:u the Mediterranean Sea (Jimenez et al., 2016; Kersting et al., 2013; conditions and to assess baseline expression levels in the natural en Rubio-Portillo et al., 2016). These events may have contributed to vironment. Further, hsp70 expression was assessed in nubbins/ polyps shaping physiological outputs of this species in response to tempera experimentally subjected to a short-term heat stress exposure (2 hat ture, likely diminishing the natural capacity of the species to recover 32 °C) and after different periods of post-stress recovery (0-24 hat (Kersting et al., 2013). The comparatively high(er) in sit:u hsp70 baseline 17 °C). Coral species with higher in siru hsp70 baseline levels had limited levels displayed by C. caespitosa may render this species less responsive (B. europaea) or no evident (C. caespitosa) hsp70 induction by heat to additional stress(ors). lndeed, no further hsp70 up-regulation during stress. This is a well -known phenomenon, common to several in post-stress recovery was observed, suggesting a limited ability of this vertebrates inhabiting shallow-water environments (Franzellitti and species to further adjust its physiology to increasing temperatures. On Fabbri, 2005; Tomanek, 2010), and has been termed "constitutive gene the whole, both physiological (Rodolfo-Metalpa et al., 2006b, 2006a) frontloading" (Barshis et al., 2013). The occurrence of a minimal con and transcriptional (present study) outcomes Support the finding of C. stitutive hsp70 expression is likely part of the strategy loaded by these caespitosa living close to its upper thermal tolerance limit within the organisms to cope with frequently encountered environmental stress Mediterranean Sea (Rodolfo-Metalpa et al., 2006b). Considering that C. conditions (Fabbri et al., 2008; Morris et al., 2013). This strategy is caespitosa is listed as endangered by the IUCN (Otero et al., 2017), these thought to confer physiological resilience by means of faster reaction at findings may contribute to the growing body of knowledge on the the protein level during transient heat stress (Bay and Palumbi, 2017; vulnerability of this species towards projected sea temperature rise. Bellantuono et al., 2012; Ruiz-jones and Palumbi, 2017). Previous Although in sit:u hsp70 baseline expression was not significantly studies highlighted a frontloading behavior of hsp70 transcripts through different amongst the azooxanthellate species, different temporal pro reciprocal transplanting of Acropora hyacinrus nubbins between sites files were observed after heat shock exposure. Based on the magnitude experiencing distinct temperature regimes (Barshis et al., 2013; Bay and of hsp70 induction in heat-shock experiments, the following ranking of Palumbi, 2017), or assessing the influence of depth-related basal ex heat sensitivity can be defined: A. calycularis > C. inomata > L. pru pression profiles on hsp70 induction in P. verrucosa (Poti et al., 2017). In voti. This ranking agrees with the relative stress tolerances of the species agreement with these observations, in this study the most remarkable predicted from variations of growth, reproduction, and population dy differences in the hsp70 responses were observed between zoox namics across environmental gradients (Carosclli et al., 2017, 2016a; anthellate and azooxanthellate species, suggesting that symbiosis may 2016b) (Fig. 1). Leptosammia pruvoti appears the less sensitive amongst play an important role in modulating stress response capabilities of the the azooxanthellate species investigated in this study, showing a very coral hosts. Specifically, zooxanthellate corals showed higher in situ Ilm ited increase of hsp70 expression only at 2 h post-stress recovery. hsp70 baseline expression than the azooxanthellates, suggesting that Indeed, this species showed unaffected physiological outputs along this might be consequence of the symbiosis that is arguably highly extended latitudinal thermal and solar irradiance gradienrs (Airi et al., susceptible to the effects of thermal Stress. Furthermore, among zoox 2017; Caroselli et al., 2012a, 2011; Goffredo et al., 2007). A. calycularis anthellate species, C. caespitosa showed higher in sit:u hsp70 baseline resuilted the most sensitive azooxanthellate species, showing the highest levels than B. europaea, and remarkably different post heat-stress hsp70 fold changes, in agreement with the relevant physiological effects transcriptional profiles were observed between these two species. Al of hypertermic stress observed in this species (Movilla et al., 2016; though composition of Symbiodinium clades hosted by the two species Prada et al., 2017). Compared to L. pruvoti, A. calycularis seems parti was not assessed in this study, previous reports on the Tyrrhenian Sea cularly sensitive to high summer temperatures, displaying a relevant showed that either ß. europaea or C. caespitosa harbored clade 82, while increase in tissue mortality that probably made the corals more sus clade A was found only in B. europaea (Barbrook et al., 2006; Meron ceptible to the detrimental effects of further stress events (Prada et al., et al., 2012). Physiologically, the "temperate" clade A is thought to 20ll7). Notably, A. calycularis maintained significant hsp70 up-regula confer potential resistance to short-term increases in temperature tion up to 24 h post-stress recovery, which may suggest a slow reactivity (Rodolfo-Metalpa et al., 2006a). Therefore, this finding may explain the or a partial temperature-dependent impairment of the cellular ma more effective hsp70 induction by heat stress displayed by B. europaea. chinery at the basis of its heat stress response, as previously observed in Besides the putative influence of symbiosis, peculiar life history traits the temperature-sensitive oyster Ostrea edulis exposed to near-lethal and adaptive featu res of these species may have a role in determining temperature increases under a similar exposure scheme (Piano et al., their observed hsp70 responses. Being sympatric species, B. europaea 2004). and C. caespitosa have a widely overlapping habitat distribution; hence, differences with regard to different environmental exposure do not 5. Conclusions serve as an explanation for the observed species-specific responses. We may hypothesize a relationship between stress responsiveness at the Data reported in this study indicate that transcriptional responses of transcriptional Jevel and colonial (C. caespitosa) vs solitary (B. europaea) the hsp70 gene align with previously reported relative differences in coral morphotype. More generally, coral morphology is thought to in stress susceptibility of the coral species investigated. Although varia fluence flow regimes and thermal fluxes at the tissue-water interface bility in environment requirements is considered a pivotal factor in (Jimcncz et al., 2011; Nakamura and Van Wocsik, 2001), tissuc thick detcrmining thc dcgrec of strcss rcsilicncc in shallow-watcr spccics, our ness and availability of resources (Loya et al., 2001), and to affect di results suggest that trophic strategy and morphology may play an im versity and light-absorbing properties of the zooxanthellae symbionts portant role in shaping coral resilience and physiological plasticity, as (Enrfquez et al., 2005; Yost et al., 2013). A recent study modeled the previously hypothesized (Samorl et al., 2017; Seveso et al., 2018). relationships between allometric scaling and rates of heating/ cooling in Combin ing baseline hsp70 expression and heat stress induction allowed coral microenvironments as a result of physical and geometric con discriminating the potential of Stress responses between zooxanthellate straints on body size and shape, and showed that, under similar water and azooxanthellate species, as weil as, within the different trophic flow and irradiance conditions, smaller corals are subjected to com strategies, between coloniaJ vs solitary corals. Whether scientific evi paratively higher stress Jevels than bigger corals (Ong et al., 2017). dences are calling into question the rote of symbiosis as a buffer to· Consequently, during the heat treatments the (smaller) solitary mor wards the adverse effects of environmental stressors (Ellison et al., photype (B. europaea) investigated in this study may have experienced 2017; Levin et al., 2016; Schwarz et al., 2008), the relevance of coral 451 morphology in shaping holobiont response to environment va riability is Javad, F., Karampelias, M., Kibenge, F„ Klbenge, M., Kumps, C., Lambertt, 1., underestimated. In th is light, results of this study Jet argue that bigger Lrunmens, T., Markey, A., Messiaen, P., Mets, E.J Morai.s, S., Mudarra-Rubio, A., (colonial) morphotype may represent a winning physiological trait to Nakiwala, J., Nelis, H., Olsvlk, P.A., Pf!rez·Novo, C., Plusquin, M., Remans, T., Rihani, A., Rodrlgues·Santos, P„ Rondou, P„ Snndcrs, R., Sdunlcl t·Bleek, K., Skovgaard, K., promote stress tolerance and adaptation in a global warming scenario. Smeets, K., Tabera, L, Toegel, S„ Van Acker, T., Vnn Den Broeck, W„ Van Der Future studies incorporating further species shouJd assess whether Meulen, J., Van Gele, M., Van Peer, G„ Van Poucke, M., Van Roy, N., Vergult, S., hsp70 expression may serve as a hallmark of coral trophic Status and Wauman, J ., Tshuikina-Wiklander, M„ Willen1S, E., Zaecara, S., Zeka, F., Vandesompele, J„ 2013. The need for transparency and good practiees in lhe qPCR morphological constraints. Understanding these molecular processes literature. 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Caroselli, E., Mattioli, G., Levy, 0., Falini, G„ Oubinsky, Z., Goffredo, S., 2012a. lnferred No competing interests declared. colcification rate of a Mediterrane.in azooxnnthe:llate coral is uncoupled with sea surface tcmperature along an 5• 1a1itudinal gradlcnl. Front. Zool. 9, 32. hups://doi org/10.1186/1742·9994 9·32. Acknowledgements Caroselli, E., Nanni, V., Levy, 0., Falinl, G., Oublnsky, Z., Goffredo, s., 2015b. l.atllud lnal varinlions in biometry and popula1lon densl1y or a mediterranean soli1ary coral. This work is part of the Master Degree Thesis in Marine Biology of Urnnol. Oeeanogr. 60, 1356-1370. hnps: 'doi.org 10.1002/lno.10100. Carosclll, E., Prada, F., Pa.squini, L , Marzano, F.N., Zaccantl, F., Falini, G., Levy, 0., D.C. The research was supported by the ltalian Ministry of Research and Oubinsky, Z., Goffredo, S„ 2011. 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