Anne-Leila Meistertzheim
email: anne-leila.meistertzheim@obs-banyuls.fr

Xavier Pochon

Susanna A Wood

Jean-François Ghiglione

Laetitia Hédouin

Development of a quantitative PCR-High Resolution Melting assay for absolute measurement of coral-Symbiodiniaceae associations and its application to investigating variability at three spatial scales

Keywords: Absolute quantification, Endosymbionts, Holobiont, Symbiosis

Previous molecular studies have shown that many corals host a dynamic consortium of dominant and background populations of Symbiodiniaceae genotypes with putatively distinct physiological traits. In the present study, we developed a quantitative PCR assay combined with High Resolution Melting analysis (qPCR-HRM) to distinguish which Symbiodiniaceae clades are present in a sample. Because the qPCR-HRM used in isolation yielded identical melt profiles for both clades A and C, this analysis was then coupled with further specific qPCR assays to enable the absolute quantification of all Symbiodiniaceae clades and host cells. When the assays were applied to in hospite samples, they had an absolute quantification level corresponding to one coral embryo of two cells and 1,000 symbiont cells. The assays were successful on coral fragments from twelve species (eight families). We then used the qPCR-HRM assay in an ecological survey of Acropora pulchra at different spatial scales (within colony, between colonies and between sites). Differences in abundance and composition of Symbiodiniaceae clades in A. pulchra were observed at all spatial scales, suggesting that various environmental factors drove changes in Symbiodiniaceae assemblages among and within coral colonies.

The qPCR-HRM assay developed in this study is a relatively simple, cost-effective and reproducible tool that can be used to accurately differentiate and quantify endosymbiont Symbiodiniaceae clades in coral in the field. This will provide new insights into coral-symbiont shuffling mechanisms and the resilience of coral colonies to environmental stressors.

Introduction

Coral reefs provide niches for 25% of all marine species but occupy only 0.2% of the world's ocean surface area, and are therefore important hot spots of biodiversity (Reaka-Kudla 1997; [START_REF] Spalding | New estimates of global and regional coral reef areas[END_REF][START_REF] Spalding | World Atlas of Coral Reefs[END_REF]. In nutrient poor environments, coral reefs depend on obligate symbiotic associations between corals and photosynthetic unicellular dinoflagellates in the family Symbiodiniaceae. This family is currently classified into nine divergent lineages, hereafter referred to as clades A to I (sensu [START_REF] Pochon | A new Symbiodinium clade (Dinophyceae) from soritid foraminifera in Hawai'i[END_REF], of which seven have recently been elevated to genus level [START_REF] Lajeunesse | Systematic revision of Symbiodiniaceae highlights the antiquity and diversity of coral endosymbionts[END_REF], and six are found in symbiosis with scleractinian corals (A-D, F and G;[START_REF] Gates | Clade D Symbiodinium in Scleractinian Corals: A "Nugget" of Hope, a Selfish Opportunist, an Ominous Sign, or All of the Above?[END_REF]Cooper et al. 2011a). Each Symbiodiniaceae clade encompasses significant within-clade genetic diversity [START_REF] Pettay | Long-range dispersal and high-latitude environments influence the population structure of a "stress-tolerant" dinoflagellate endosymbiont[END_REF][START_REF] Thornhill | Population genetics of reef coral endosymbionts (Symbiodinium, Dinophyceae)[END_REF] corresponding to multiples species or types [START_REF] Coffroth | Genetic diversity of symbiotic dinoflagellates in the genus Symbiodinium[END_REF]. Experimental and field-based studies of coral bleaching events have shown that the physiology of the coral host is influenced by the genotypes of associated Symbiodiniaceae [START_REF] Rowan | Coral Bleaching:Thermal adaptation in reef coral symbionts[END_REF][START_REF] Berkelmans | The role of zooxanthellae in the thermal tolerance of corals: a "nugget of hope" for coral reefs in an era of climate change[END_REF]Stat et al. 2006;[START_REF] Howells | Coral thermal tolerance shaped by local adaptation of photosymbionts[END_REF][START_REF] Howells | Spatial and temporal genetic structure of Symbiodinium populations within a common reef-building coral on the Great Barrier Reef[END_REF]. For example, corals dominated by clade A symbionts were described as more prone to parasitism [START_REF] Gates | Clade D Symbiodinium in Scleractinian Corals: A "Nugget" of Hope, a Selfish Opportunist, an Ominous Sign, or All of the Above?[END_REF]), whereas associations with clade C improved overall growth [START_REF] Little | Flexibility in Algal Endosymbioses Shapes Growth in Reef Corals[END_REF][START_REF] Cantin | Juvenile corals can acquire more carbon from high-performance algal symbionts[END_REF], and with type D1a (Durusdinium trenchii sensu LaJeunesse et al. 2018) thermo-tolerance was enhanced [START_REF] Baker | Symbiont diversity on coral reefs and its relationship to bleaching resistance and resilience[END_REF][START_REF] Gates | Clade D Symbiodinium in Scleractinian Corals: A "Nugget" of Hope, a Selfish Opportunist, an Ominous Sign, or All of the Above?[END_REF].

The association between a coral and several Symbiodiniaceae clades, often results in the presence of one dominant clade together with several background clades. The formation of partnerships between a coral and dominant Symbiodiniaceae clades often appears stable [START_REF] Thornhill | Reef endemism, host specificity and temporal stability in populations of symbiotic dinoflagellates from two ecologically dominant Caribbean corals[END_REF][START_REF] Lajeunesse | Host-symbiont recombination versus natural selection in the response of coral-dinoflagellate symbioses to environmental disturbance[END_REF], and possibly driven by environmental conditions [START_REF] Bongaerts | Sharing the slope: depth partitioning of agariciid corals and associated Symbiodinium across shallow and mesophotic habitats (2-60 m) on a Caribbean reef[END_REF][START_REF] Tonk | Symbiodinium (Dinophyceae) diversity in reef-invertebrates along an offshore to inshore reef gradient near Lizard Island, Great Barrier Reef[END_REF][START_REF] Kennedy | Symbiodinium biogeography tracks environmental patterns rather than host genetics in a key Caribbean reef-builder, Orbicella annularis[END_REF]. However, the ability of corals to associate with multiple symbiont assemblages has been shown to be more widespread than previously reported [START_REF] Silverstein | Specificity is rarely absolute in coral-algal symbiosis: implications for coral response to climate change[END_REF].

The fragile symbiosis between corals and zooxanthellae is undergoing drastic changes due to shifting environmental conditions [START_REF] Hughes | Climate change, human impacts, and the resilience of coral reefs[END_REF][START_REF] Hoegh-Guldberg | Coral reefs under rapid climate change and ocean acidification[END_REF]). Rising seawater temperatures has led to an increase in stress-induced symbiosis breakdowns, also known as coral bleaching. Several massive coral bleaching events have been reported, resulting in a worldwide coral loss of 16% in 1998 [START_REF] Wilkinson | Status of Coral Reefs of the World 2000[END_REF]. The most recent event was related to intense drought linked to El Niño (2015/2016), which resulted in the third worst worldwide mass bleaching event (National Oceanic and Atmospheric Administration [NOAA] Coral ReefWatch program 2016). The impact of this event is still incomplete but it is believed that it was the most severe coral bleaching event on record in the Great Barrier Reef [START_REF] Hughes | Global warming and recurrent mass bleaching of corals[END_REF]NOAA Coral ReefWatch program 2017). During abnormally high seawater temperatures, certain coral species are more resistant to bleaching than others, and some bleached corals can recover (e.g. [START_REF] Jones | A community change in the algal endosymbionts of a scleractinian coral following a natural bleaching event: field evidence of acclimatization[END_REF]. This observed resilience may be due to the capability of some corals to 'switch' their symbionts (the uptake of more tolerant symbionts from the surrounding environment) or 'shuffle' their existing assemblage composition (background taxa may become abundant in higher temperatures; [START_REF] Baker | Flexibility and specificity in coral-algal symbiosis: Diversity, ccology , and biogeography of Symbiodinium[END_REF][START_REF] Baker | Symbiont diversity on coral reefs and its relationship to bleaching resistance and resilience[END_REF]LaJeunesse et al. 2009).

Few studies have investigated the impact of changes in background symbiont composition and density on coral stress resistance (e.g. Cunning andBaker 2012, 2014), and how this composition shifts after bleaching events [START_REF] Cunning | Excess algal symbionts increase the susceptibility of reef corals to bleaching[END_REF][START_REF] Mcginley | Symbiodinium spp. in colonies of eastern Pacific Pocillopora spp. are highly stable despite the prevalence of low-abundance background populations[END_REF]Cunning et al. 2015a[START_REF] Cunning | Patterns of bleaching and recovery of Montipora capitata in Kāne'ohe Bay, Hawai'i, USA[END_REF][START_REF] Bay | Recovery from bleaching is mediated by threshold densities of background thermo-tolerant symbiont types in a reef-building coral[END_REF]. It has been shown that the Symbiodiniaceae taxonomic composition may change within a coral colony [START_REF] Ulstrup | Inter-polyp genetic and physiological characterisation of Symbiodinium in an Acropora valida colony[END_REF][START_REF] Kemp | A microsampling method for genotyping coral symbionts[END_REF][START_REF] Kemp | Community dynamics and physiology of Symbiodinium spp. before, during, and after a coral bleaching event[END_REF]. However, variation in absolute abundances (symbionts normalized to host parameters, such as units of DNA or cell numbers) of the nine Symbiodiniaceae clades remains unexplored. Development of a method to accurately measure absolute abundances of all nine Symbiodiniaceae clades would enable significant advancements in our understanding of the functional role of symbiont assemblages in coral symbiosis [START_REF] Cunning | Not just who, but how many: the importance of partner abundance in reef coral symbioses[END_REF].

Quantitative PCR (qPCR) allows the description of the relative abundance of different symbiont clades, based on the number of copies of specific DNA loci within a sample [START_REF] Ulstrup | Geographic and habitat partitioning of genetically distinct zooxanthellae (Symbiodinium) in Acropora corals on the Great Barrier Reef[END_REF][START_REF] Loram | Molecular quantification of symbiotic dinoflagellate algae of the genus Symbiodinium[END_REF][START_REF] Mieog | Real-time PCR reveals a high incidence of Symbiodinium clade D at low levels in four scleractinian corals across the Great Barrier Reef: implications for symbiont shuffling[END_REF]). It has previously enabled the effective tracking of symbiont communities dynamics from five symbiont clades (A, B, C, D and F), that were standardized to host cells [START_REF] Cunning | Excess algal symbionts increase the susceptibility of reef corals to bleaching[END_REF], Cunning et al., 2015a, Mieog et al. 2009). It has also enabled detection of low-abundance Symbiodiniaceae genotypes associated with corals [START_REF] Silverstein | Specificity is rarely absolute in coral-algal symbiosis: implications for coral response to climate change[END_REF][START_REF] Rouzé | Symbiodinium clades A and D differentially predispose Acropora cytherea to disease and Vibrio spp. colonization[END_REF]) that were previously thought to harbor only a single dominant clade [START_REF] Goulet | Most scleractinian corals and octocorals host a single symbiotic zooxanthella clade[END_REF]; but see Baker and[START_REF] Baker | Multiple symbiotic partnerships are common in scleractinian corals, but not in octocorals: Comment on Goulet (2006)[END_REF][START_REF] Mieog | Real-time PCR reveals a high incidence of Symbiodinium clade D at low levels in four scleractinian corals across the Great Barrier Reef: implications for symbiont shuffling[END_REF]). However, absolute quantification of all Symbiodiniaceae clades had not yet been achieved because of; i) the lack of a robust and efficient DNA extraction method applicable to a wide range of sample types (cells/polyps, colonies, and replicate), ii) the inability of qPCR assays to target all clades (i.e. including clades G-I) and the absence of an internalcontrol for identifying missing clades (i.e., a family-Symbiodiniaceae target), and iii) the lack of qPCR amplification efficiency measurements amongst clades and single host cells.

A further benefit of qPCR is the ability to include High Resolution Melt curve to differentiate PCR products with different sequences. Two sequences containing at least one mutation generate fragments that rapidly denature and rejoin after PCR to form two (high-melting) homoduplexes and two (lowmelting) heteroduplexes (base-pairing mismatches). The melting properties of these double-stranded PCR products are monitored through the release of a saturating fluorescent double stranded DNA binding dye as temperatures are increased, thus forming different melting curve shapes [START_REF] Wittwer | High-Resolution Genotyping by Amplicon Melting Analysis Using LCGreen[END_REF]. Homozygous variants are therefore distinguishable from each other through differences in their melting temperature. We previously demonstrated that the identification of unknown specimens is undertaken by comparing these specimens to a set of reference melting curves [START_REF] Meistertzheim | High Resolution Melting Analysis for fast and cheap polymorphism screening of marine populations[END_REF].

The aim of this study was to develop an accurate, fast and highly reliable qPCR-High Resolution Melting (qPCR-HRM) assay that could be coupled with further qPCR assays to distinguish and quantify the nine Symbiodiniaceae clades in a wide range of coral taxa. For this purpose, we designed seven new cladespecific primer sets and one family-Symbiodiniaceae primer set, which were tested on a range of cultures and pure DNA samples from each of the nine existing Symbiodiniaceae clades. Additionally, two coralspecific (actin and nuclear ribosomal) primer sets were designed and used on Symbiodiniaceae-free embryos of Acropora pulchra to produce host standard curves for the quantification of coral cells. We validated our assay on colony fragments from twelve different species representing eight different families, and undertook an ecological survey of A. pulchra at different micro-and macro-scales (within colonies, between colonies and among sites).

Material and methods

Primer development

Alignment of 135 sequences of the nuclear large sub-unit ribosomal (28S) rRNA gene, including the 5' regions of the 5.8S and Internal Transcribed Spacer 2 (ITS2), were performed using MEGA6 [START_REF] Tamura | MEGA6: Molecular Evolutionary Genetics Analysis Version 6.0[END_REF]. Seven clade-specific forward/reverse primer sets were designed using Primer Express v2.0 software (Applied Biosystems). The primers targeted an approximately 70-150 nucleotides [nt] region, one of the forward or reverse primer being located near the 3' end of the 5.8S rRNA gene or the 5' end of the 28S rRNA gene, and the corresponding reverse or forward primer being located in the ITS2 region (see Table 1). We also used previously published primers, including SYM_VAR_5.8S2 [START_REF] Hume | Symbiodinium thermophilum sp nov., a thermotolerant symbiotic alga prevalent in corals of the world's hottest sea, the Persian/Arabian Gulf[END_REF], and two actin primer sets specific to clades C and D [START_REF] Cunning | Excess algal symbionts increase the susceptibility of reef corals to bleaching[END_REF]. Host-specific (coral) primers were designed using alignments of the actin and nuclear 5.8S rDNA genes from GenBank (Table S1).

HRM analysis was used to quantify the number of clades present in each sample. This requires the amplification of a variable 80-nt partial sequence of the 5.8S gene using the forward SYM_VAR_5.8S2 and reverse Symb_R primer, hereafter referred to as the family primer pair 'Symb', which targets conserved flanked areas amplifying the nine different Symbiodiniaceae clades (Table 1; Figure S1).

There are between one to ten mutations among clades in this gene (Table S2), which allows the distinction between them based on melting curve shape.

Absolute quantification assay development

Primer specificity was verified using DNA isolated from Symbiodiniaceae cell cultures in their vegetative state, from DNA collections of clades G-I (Table S3), and embryos of A. pulchra. Cells from Symbiodiniaceae-free embryos at division 2 and 4 cells were obtained following the procedure described in [START_REF] Hédouin | Hyposalinity stress compromises the fertilization of gametes more than the survival of coral larvae[END_REF], using A. pulchra colonies that were collected in September 2012 on the fringing reefs of Moorea Island (17° 30' S, 149° 50'W, French Polynesia, Society Archipelago, Figure 1 A-B).

We tested a new and automated extraction method, applicable to both coral embryos and polyps, which yields consistent DNA quantity between replicates and allowed successful DNA extraction from 2 cells for corals embryos. Symbiodiniaceae cultures, embryos and samples from coral colonies were lysed by mechanic action using the Y Matrix on a FastPrep Instrument (MP Biomedical, USA). Samples were incubated with proteinase K (57°C, 1 hr). DNA was then isolated using a Maxwell® Blood DNA Purification Kit LEV (Promega, USA) on a Maxwell 16 MDx Instrument (Promega). DNA quality was assessed by electrophoresis on a 1% agarose gel. Total genomic DNA concentration were measured using spectrophotometry (Nanodrop ND-1000, Thermo Fisher scientific Inc., MA, USA).

Standard curves of Symbiodiniaceae and host cells were constructed to allow absolute quantification (Table S2). Living Symbiodiniaceae cells were quantified using a Scepter Handheld Automated Cell Counter system (Millipore, USA) combined with Scepter Pro software (Millipore). Automated cell counts were compared to cell counts obtained using a hemacytometer under a light microscope. DNA was extracted for each concentration as described previously. Standard curves were run in triplicate with cell concentrations of Symbiodiniaceae clades of 1×10 3 to 3×10 6 cells per DNA extraction (clades A-F), and using DNA concentrations ranging between 0.5 and about 40 ng DNA µL -1 (clades G, H and I).

Two standard curves for the quantification of host-cells were run in triplicate using Symbiodiniaceaefree embryos with an increasing concentration of 2 to 400 cells per DNA extraction. Acropora pulchra were in their first and second division stages, and concentrations were determined by binocular microscopy.

Quantitative PCR reactions were performed in duplicate in a 10 µL volume containing genomic DNA, 1× High Resolution Melting master mix (Roche Diagnostics, USA) and 0.3 µM of each primer. The amplification protocol consisted of 10 min pre-incubation at 95ºC followed by 40 cycles of 95ºC for 15 s (ramp rate, 4.4 ºC. s -1 ), touchdown annealing from 65 to 53 ºC for 15 s (ramp rate, 2.2 ºC s -1 ), and extension at 72ºC for 20 s (s -1 ramp rate, 4.4 ºC. s -1 ). The qPCR reactions were performed in 384-well plates using a LightCycler®480 Instrument (Roche Diagnostics, USA). After qPCR amplification, genotyping using HRM was undertaken. This consisted of a denaturing step at 95 ºC for 1 min, followed by a hybridization step at 40 ºC for 1 min. Melting curves were then constructed by ramping from 65 to 95ºC at 0.02ºC s -1 , taking 25 acquisitions of fluorescence measure per each degree centigrade. A negative (no-template) control was included in the PCR to detect potential DNA contamination or any formation of primer dimers. Primer specificity was evaluated using qPCR analysis, gel electrophoresis and bidirectional sequencing of each positive qPCR product.

For each qPCR reaction, the crossing point (Cp; corresponding to the cycle threshold [Ct]) was determined using the "second derivative max method" implemented in the LightCycler®480 v.1.5.0 Software (Roche Diagnostics). The threshold for Ct calculation and normalization regions (leading and trailing ranges) for qPCR-HRM analysis were defined for each Symbiodiniaceae clade and host locus using serial dilutions of DNA and standard curves. Only successfully amplified samples with a Cp value within the linear part of the standard curves were considered for further analysis [START_REF] Bustin | The MIQE guidelines: minimum information for publication of quantitative real-time PCR experiments[END_REF]. Individual qPCR efficiencies (E) of the Symbiodiniaceae clades and host reference genes were calculated using the formula: E=10 (-1/slope) , as described by [START_REF] Yuan | Statistical analysis of real-time PCR data[END_REF]. All primers had a qPCR efficiency between 1.84 and 2.02 (Table 1).

Conversion of quantification cycle values

Cp into relative quantities 𝑄 𝑐𝑙𝑎𝑑𝑒 𝑖 was undertaken for each DNA concentrations of the standard curves i using clade-specific primer pair using the modified equation described by [START_REF] Pfaffl | A new mathematical model for relative quantification in real-time RT-PCR[END_REF] without normalization by a reference gene:

𝑄 𝑐𝑙𝑎𝑑𝑒 𝑖 = 𝐸 𝑐𝑙𝑎𝑑𝑒 𝑖 ∆𝐶𝑡,𝑐𝑙𝑎𝑑𝑒 𝑖 , (arbitrary unit)
where 𝐸 𝑐𝑙𝑎𝑑𝑒 𝑖 is the amplification efficiency of the clade-specific primer couple i and ΔCt, cladei =

𝐶𝑡 𝑐𝑙𝑎𝑑𝑒 𝑖 (calibrator) -𝐶𝑡 𝑐𝑙𝑎𝑑𝑒 𝑖 (sample).
The calibrator consisted of the maximal concentration of each Symbiodiniaceae clade included in the linear part of the standard curves.

The 𝑄 𝑐𝑙𝑎𝑑𝑒 𝑖 was calculated for each DNA concentrations in nanograms (ng) from the linear part of each standard curve i, allowing the derivation of the following equation:

𝐷𝑁𝐴 𝑐𝑙𝑎𝑑𝑒 𝑖 = 𝑓(𝑄 𝑐𝑙𝑎𝑑𝑒 𝑖 ) (ng)
Similarly, the family primer pair 'Symb' was used to amplify total DNA copies of Symbiodiniaceae in one reaction such that:

𝑄 𝑇𝑜𝑡𝑆𝑦𝑚𝑏 = 𝐸 𝑆𝑦𝑚𝑏 ∆𝐶𝑡,𝑆𝑦𝑚𝑏 , (arbitrary unit)

The 𝑄TotSymb was then calculated for each DNA concentrations from the Symbiodiniaceae standard curves, subsequently allowing for the derivation of the following equation:

𝐷𝑁𝐴 𝑇𝑜𝑡𝑆𝑦𝑚𝑏 = 𝑓(𝑄 𝑇𝑜𝑡𝑆𝑦𝑚𝑏 ) (ng)
These standard curves allowed for the concentrations of each clade i and total Symbiodiniaceae (per cell or DNA unit) of the unknown samples to be determined (Pfaffl, 2004). We considered this method as sufficiently accurate when 𝐷𝑁𝐴 𝑆𝑦𝑚𝑏 = ∑ 𝐷𝑁𝐴 𝑐𝑙𝑎𝑑𝑒 𝑖 9 𝑖=1

(ng).

The quantity of host cells was calculated using the two 𝑄 𝑟𝑒𝑓 𝑖 calculated as described above from the two host reference genes. The calculated values were plotted against the linear regression of each corresponding standard curve, where:

𝑄 𝑟𝑒𝑓 𝑖 = 𝐸 𝑟𝑒𝑓 𝑖 ∆𝐶𝑡,𝑟𝑒𝑓 𝑖 , (arbitrary unit)
Finally, the concentration of host cells was assessed by the geometric mean of the 𝑄 𝑟𝑒𝑓 𝑖 such as described by [START_REF] Hellemans | qBase relative quantification framework and software for management and automated analysis of real-time quantitative PCR data[END_REF] to normalize relative quantities with multiple reference genes:

𝑄 𝐻𝑜𝑠𝑡 = √∏ 𝑄 𝑟𝑒𝑓 𝑖 2 𝑖
The 𝑄 𝐻𝑜𝑠𝑡 was then calculated for each DNA concentrations from the host standard curves, subsequently allowing for the derivation of the following equation:

𝐷𝑁𝐴 𝐻𝑜𝑠𝑡 = f(𝑄 𝐻𝑜𝑠𝑡 ) (ng).
Finally, the linear part of each standard curve allowed for the quantification of both Symbiodiniaceae and its host using specific and family primers starting from a known quantity of DNA, such that

𝐷𝑁𝐴 𝑅𝑒𝑎𝑐𝑡𝑖𝑜𝑛 = 𝐷𝑁𝐴 𝐻𝑜𝑠𝑡 + 𝐷𝑁𝐴 𝑆𝑦𝑚𝑏 (ng)
and

𝐷𝑁𝐴 𝑅𝑒𝑎𝑐𝑡𝑖𝑜𝑛 = 𝐷𝑁𝐴 𝐻𝑜𝑠𝑡 + ∑ 𝐷𝑁𝐴 𝑐𝑙𝑎𝑑𝑒 𝑖 9 𝑖=1
(ng)

The HRM data obtained with the family-Symbiodiniaceae primers 'Symb' resulted in differences in curve shapes for each Symbiodiniaceae clade which were discriminated using the Gene Scanning

Validation test of host quantification using other coral species

We then tested the applicability of only the two coral reference genes to be applied to species other than A. pulchra because the other primer sets will target Symbiodiniaceae clades whatever the host species.

Of the 67 scleractinian species from nine different families previously described in Moorea Island Two or three colony fragments (ca. 0.5-1 cm 2 ) of the coral species were sampled by diving in several reef sites of Moorea and Tahiti Islands. The coral fragments were collected using wire cutters and clamps previously cleaned with sodium hydroxide and ethanol to avoid cross-contamination between samples.

Each fragment was collected from the tip of each coral and fixed separately in 70% ethanol.

Coral samples were washed in DNAse-free water before performing DNA extraction, as described above. DNA was diluted to 15 ng µL -1 and 30 ng was used to conduct qPCR-HRM assays. To ensure replicability and homogeneity, each plate contained DNA from a representative of the nine Symbiodiniaceae clades in duplicate, as well as Symbiodiniaceae-free coral embryos and a negative control.

Comparison of Symbiodiniaceae assemblages in Acropora pulchra at micro and macro-scale

Twenty colonies of A. pulchra were collected in September 2012 from Mahana (17°29'18"S; 149°53'7"W) and Teavaro (17°30'23"S; 149°45'53"W), two fringing reef sites in Moorea Island (Figure 1). These two sites were chosen because A. pulchra colonies were found in high abundances at both sites despite contrasting turbidity (0.12 vs 0.04 NTU in Mahana and Teavaro sites, respectively).

Turbidity, salinity, temperature, and pH were recorded using the multiparameter HORIBA U50 probe.

To examine the spatial distribution of symbiont clades within a host colony, branch fragments (approximately 0.5-1 cm 2 ) were sampled using individual sterilized wire cutters and clamps. Coral samples were collected on both the upward and downward facing sides of coral branches, exhibiting two distinct color morphs (normal [N] and slightly bleached [sB]), representing different micro-variation of the environment; Figure 1C-E). These were preserved in 70% ethanol and stored at -20°C until DNA extraction. DNA extractions were undertaken as described above. Absolute quantification by qPCR assay and HRM was undertaken in duplicate using standard curves of Symbiodiniaceae clades and A.

pulchra embryos with a no-template control in each plate.

Tests for normality and homoscedasticity on obtained DNA concentrations were performed using the Statistica software v10 (Statsoft) using Shapiro-Wilk and Levene tests. Concentration differences between coral-color fragments and sites for the number of host-cells, total Symbiodiniaceae cells, and cell counts for each specific Symbiodiniaceae clade were compared using non-parametric Kruskal-Wallis and Mann-Whitney U-tests.

Results

Quantification of symbiont and host cells

Standard curves generated from increasing Symbiodiniaceae cells ranging from 1x10 3 to 3x10 6 cells per extraction resulted in DNA ranging concentrations of 1 to 200 ng DNA µL -1 . For all clades, there was a strong correlation between the DNA concentrations and the cell numbers with correlation coefficient R 2 higher than 0.95 (e.g., for clade C R 2 =0.96 ± 0.03 SD; Figure S2A). Linear curves were obtained for host cell, with DNA concentrations gradually increasing (9 to 670 ng DNA µL -1 ) as the number of host cells per extraction rose from 2 to 400 (R 2 =0.996 ± 0.004, Figure S2B). Using qPCR analysis, quantification of the total number of Symbiodiniaceae cells was performed using the family primer pair 'Symb' with DNA extracted from 1×10 3 to 3×10 6 cells per extraction of all individual clades (Table S4).

The resulting standard curves obtained for each clade (𝐷𝑁𝐴 𝑇𝑜𝑡𝑆𝑦𝑚𝑏 = 𝑓(𝑄 𝑇𝑜𝑡𝑆𝑦𝑚𝑏 )) indicated high correlation coefficients (mean R 2 =0.96 ± 0.04; Figure 2A for clade C). The clade-specific Symbiodiniaceae primers, also had standard curves (𝐷𝑁𝐴 𝑐𝑙𝑎𝑑𝑒 𝑖 = 𝑓(𝑄 𝑐𝑙𝑎𝑑𝑒 𝑖 )) with high coefficient correlation values (mean R 2 =0.96 ± 0.04; Figure 2A for clade C and Table S4). We obtained similar specificity and efficiency (ESymb) for qPCR amplifications tested on representative samples from each of the nine clades using the family primers 'Symb' (Table S5), suggesting that these loci are conserved within Symbiodiniaceae lineages. Host standard curves, which allowed for the quantification of polyp cells, were constructed (2 to 200 cells; Figure 2B) with the highest accuracy obtained between 2 and 50 cells (R 2 =0.98 ± 0.02), corresponding to DNA concentrations ranging between 10 and 90 ng DNA µL -1 (Figure S2B). No DNA amplification was obtained from embryos cells when using primers designed for Symbiodiniaceae, or from dinoflagellates cells when using primers designed for host genes.

Duplicates produced gap values between both Ct lower than 0.5.

Reproducibility, accuracy and specificity of the High Resolution Melt curve genotyping

The HRM assay using the family locus of 5.8S rDNA for the entire genus Symbiodiniaceae allowed for correct discrimination between all clades except clades A and C when compared to the selected cell cultures and DNA collection (Table S2). Discrimination was explored by visual inspection of the melting curves (Figure 3A and 3B) and by calculating RMSE values (Figure 3C). The visual and mathematical distinction between clades was clearly visible for all Symbiodiniaceae lineages explored in this study, except for clades A and C. However, when different clades are co-occurring, the method effectively detected the presence of heteroduplexes (e.g. AC; see below). Symbiodiniaceae identification by HRM using the family-specific primer pair (Symb) on material derived from cell cultures always corroborated the results from clade-specific primers. The Tm of all analyzed samples ranged between 82°C and 84°C (Figure 3A). Seven clades were easily identifiable and the mean RMSE values for comparisons of samples from the nine clades (Figure 3C) ranged from 2.70 (±0.16) to 32.56 (±0.03), corresponding to the number of mutations (1-13, Table S3). The lowest RMSE values were obtained when comparing clades A and C (mean 0.84 ± 0.09), whose sequences differed by five mutations (Table 2). Although up to two mutations were present within the reverse (SymbR) primer region (Figure S1), similar amplification efficiencies were obtained using the family primer in the cladespecific standard curves (Figure 2), highlighting similar amplification efficiencies between clades (Table S5). No DNA amplification was obtained from our negative controls. Nine replicate reactions per clade and per experimental run, including standard curves of cell cultures and triplicate DNA samples, yielded similar Ct values (ΔCt <0.5) and similar melting curves corresponding to low RMSE for intra-and inter-run comparisons (mean values of 1.0 ± 0.3 and 2.2 ± 0.6, respectively). Similar melting curve shapes were obtained among sub-clade genotypes (Table S2) within five clades (A, B, F, G and H), with RMSE values intra-clades ranging from 0.09 to 2.12 (mean value of 1.25 ± 0.46) (Table S5).

Limits of the High Resolution Melt curve method and application to coral tissue samples

We obtained similar melting curve shapes for the nine Symbiodiniaceae clades, with DNA concentrations ranging from 100 pg to 500 ng and a mean RMSE value of 0.68 ± 0.37. It indicated that low template DNA concentration did not significant change the melting curve shapes. Melting curves obtained using experimental mixtures of pairwise clade DNA comparisons (using mixed combinations among clades A, B, C, D and F) always displayed more than two peaks, including the mixture of A and C, thus confirming the presence of two different DNA amplifications and their corresponding homoduplexes and heteroduplexes in the reaction (Figure 4A). DNA mixed in the same proportions using three, four and/or five clades always yielded melting curve shapes with three peaks regardless of the proportions used (Figure 4B), confirming the presence of three or more clades in a reaction which was further validated by three or more positive amplifications using the specific primers. However, the intensity of both peaks changed when applying variable proportions of two clades (Figure 4C).

The method assumes that the copy number of the locus targeted by the qPCR primers is the same within each clade. However, further investigations are required whether this assumption is valid and can be applied to the absolute quantification of all clades in coral samples.

Validation tests of host quantification using other coral species

For the twelve coral species, amplifications of the host DNA using the host-specific primer sets were positive regardless of the coral species investigated in this study. The Cp values of the host primer set (host genes) always fell within the constructed standard curves using A. pulchra embryos, i.e. less than 30 PCR cycles (data not shown).

Field survey of Acropora pulchra at micro and macro-scales

We analyzed a total of 40 fragments of A. pulchra collected from two sites (Mahana and Teavaro) and including two distinct color morphs. Out of the nine existing Symbiodiniaceae clades, only three (A, C, and D) were detected using the qPCR-HRM assay (Figures 5 and6). Colonies showed significantly higher concentrations of total Symbiodiniaceae DNA per total DNA in Mahana compared to Teavaro (mean of 𝐷𝑁𝐴 𝑇𝑜𝑡𝑆𝑦𝑚𝑏 ± SD = 12.1% ± 3.0%, and 7.1% ± 1.3%, respectively, Mann-Whitney U-test, p<0.001). The values were similar between the normal and slightly bleached coral fragments within a site (Figure 5). Concentrations of clade A cells were 2-fold higher in colonies from Mahana compared to Teavaro (mean 6.5% ± 1.6% and 2.7% ± 0.4%, respectively; Mann-Whitney U-test, p<0.001). Clade C cells were identified almost exclusively in colonies from Teavaro at a mean concentration of 7% ± 1%, except for two colonies in Mahana with medium color loss (Figure 6). For these two colonies, fragments collected from a branch (above vs below) harbored different proportions of Symbiodiniaceae (8.2% vs 5.4%, 0% vs 3.0% and 5.6% vs 2.6%, for clades A, C and D, respectively, Figure 6). In contrast, clade D cells were detected exclusively from Mahana colonies (mean 4.8% ± 2.6%).

Discussion

Quantitative PCR-High Resolution Melt assay

This study developed a new qPCR-HRM assay to explore Symbiodiniaceae clades present in a coral fragment smaller than 1 cm 2 . This assay was coupled with further clade and host specific assays and allowed, for the first time, the absolute quantification of the proportions of each Symbiodiniaceae clade in coral tissues normalized by the number of host cells. This quantification was based on standard curves generated using Symbiodiniaceae-free coral cells and representative DNA or cultures from each of the nine existing clades, the latter being haploid in vegetative stage in both cultured and in hospite [START_REF] Santos | Molecular genetic evidence that dinoflagellates belonging to the genus Symbiodinium Freudenthal are haploid[END_REF]. The method assumes that copy number of the locus targeted by the qPCR primers is the same in the cells used as the standard curve template and the cells present in the unknown sample (within each clade). This allows for the establishment of a correlative relationship between the number of living cells in each clade and qPCR values. However, additional research is required to confirm this assumption.

The qPCR-HRM assay developed in the present study provides a simple way to identify which clades are present in mixed environmental samples. Granados-Cifuentes and Rodriguez-Lanetty ( 2011)

showed that HRM is a more effective option, than relatively time-consuming molecular fingerprinting methods such as Denaturing Gradient Gel Electrophoresis for identifying mixed-clade combinations.

Our results demonstrated that HRM is able to identify each Symbiodiniaceae clade in isolation but failed to differentiate clades A and C due to their identical melting curves. Two amplicons can have the same melting behavior despite sequences not being exactly the same, e.g., due to an erasing effect caused by two mutations (e.g. T to G and G to T) [START_REF] Druml | High resolution melting (HRM) analysis of DNA -Its role and potential in food analysis[END_REF]. However, the present study also demonstrated that when different clades are mixed together, including the mixing of A and C, heteroduplexes are always produced which are easily detectable by HRM because they alter the shape of the melting curves [START_REF] Wittwer | High-Resolution Genotyping by Amplicon Melting Analysis Using LCGreen[END_REF]. Our HRM results were also corroborated by clade-specific qPCR amplifications. By combining HRM with qPCR, we gained insights into the absolute quantification of the coral polyp and its symbiont cladal content. Quantitative PCR analyses has previously been described as a powerful method for quantifying Symbiodiniaceae clades A-F [START_REF] Loram | Molecular quantification of symbiotic dinoflagellate algae of the genus Symbiodinium[END_REF][START_REF] Mieog | Real-time PCR reveals a high incidence of Symbiodinium clade D at low levels in four scleractinian corals across the Great Barrier Reef: implications for symbiont shuffling[END_REF][START_REF] Bay | Recovery from bleaching is mediated by threshold densities of background thermo-tolerant symbiont types in a reef-building coral[END_REF][START_REF] Rouzé | Symbiodinium clades A and D differentially predispose Acropora cytherea to disease and Vibrio spp. colonization[END_REF][START_REF] Rouzé | An updated assessment of Symbiodinium spp. that associate with common scleractinian corals from Moorea (French Polynesia) reveals high diversity among background symbionts and a novel finding of clade B[END_REF]. These studies quantified the relative proportion of PCR amplicons from each symbiotic clade, standardizing their assays using a single host gene. However, there was no in-depth assessment of the potential effect additional clades (i.e., G, H, and I) may have on the overall assay. Furthermore, differential efficiencies of PCR amplification among clades and host cells were not taken into account possibly leading to an underestimation of clade proportions. These assays also lacked total Symbiodiniaceae cell internal controls. The assay described in the present study quantifies host cells through amplification of two conserved reference host genes, and we showed that this could be applied to twelve coral species representing eight different coral families. Using specific and family primers of the nine Symbiodiniaceae clades and two host genes to construct standard curves of symbiotic and host cells, our method quantifies the total abundance of Symbiodiniaceae and each specific clade composition, normalized per host parameters, in terms of DNA values (in nanograms) or cell numbers, as previously recommended [START_REF] Cunning | Not just who, but how many: the importance of partner abundance in reef coral symbioses[END_REF].

Limitations

Using high concentrations of DNA can lead to misidentification of clades when using qPCR-HRM assays (Granados-Cifuentes and Rodriguez-Lanetty 2011). In the present study, the upper limit of DNA templates for the assay was not reached with maximal values of DNA for embryos at 670 ng.µL -1 and Symbiodiniaceae at 200 ng.µL -1 . We also obtained accurate and reproducible results with 30 ng of total DNA extracted from each coral fragment. The lower limit of the method corresponded to one embryo of two cells, and to 10 3 cells of Symbiodiniaceae. This limit is lower than population sizes measured within planulae (7.4.10 4 in 1 mm long; Marshall 1932) or within individual fragments of coral colony (~10 6 cells cm -2 ; [START_REF] Fitt | Seasonal patterns of tissue biomass and densities of symbiotic dinoflagellates in reef corals and relation to coral bleaching[END_REF]. This method is therefore particularly useful for accurately detecting background Symbiodiniaceae clades within a sample. Unlike High-Throughput Sequencing (HTS) methods [START_REF] Arif | Assessing Symbiodinium diversity in scleractinian corals via next-generation sequencing-based genotyping of the ITS2 rDNA region[END_REF][START_REF] Edmunds | Persistence and Change in Community Composition of Reef Corals through Present, Past, and Future Climates[END_REF][START_REF] Quigley | Deep-sequencing method for quantifying background abundances of Symbiodinium types: Exploring the rare Symbiodinium biosphere in reef-building corals[END_REF][START_REF] Thomas | Exploring Symbiodinium diversity and host specificity in Acropora corals from geographical extremes of Western Australia with 454 amplicon pyrosequencing[END_REF][START_REF] Boulotte | Exploring the Symbiodinium rare biosphere provides evidence for symbiont switching in reefbuilding corals[END_REF][START_REF] Hume | An improved primer set and amplification protocol with increased specificity and sensitivity targeting the Symbiodinium ITS2 region[END_REF], our assay is unable to provide information on the total genetic diversity of within-clade assemblages. Nevertheless, HTS methods are relatively costly and time-consuming compared to the qPCR-HRM method presented here, which requires fewer processing steps and produces results within hours and at relatively low cost. This method may effectively complement HTS studies when dealing with large numbers of small individual samples [START_REF] Cousins | Comparison of a High Resolution Melting (HRM) Assay to Next Generation Sequencing for Analysis of HIV Diversity[END_REF][START_REF] Cristescu | From barcoding single individuals to metabarcoding biological communities: towards an integrative approach to the study of global biodiversity[END_REF].

Endosymbiotic Symbiodiniaceae genotypes composition of Acropora pulchra at different spatial scales

The ecological role of dominant Symbiodiniaceae genotypes in coral host physiology and stress tolerance has been extensively studied [START_REF] Rowan | Intraspecific diversity and ecological zonation in coral-algal symbiosis[END_REF][START_REF] Baker | Flexibility and specificity in coral-algal symbiosis: Diversity, ccology , and biogeography of Symbiodinium[END_REF][START_REF] Coffroth | Genetic diversity of symbiotic dinoflagellates in the genus Symbiodinium[END_REF][START_REF] Putnam | Endosymbiotic flexibility associates with environmental sensitivity in scleractinian corals[END_REF]. However, there is very limited understanding of the natural variability in Symbiodiniaceae cladal abundance distribution in hospite, due to micro-environmental conditions [START_REF] Wangpraseurt | The in situ light microenvironment of corals[END_REF][START_REF] Wangpraseurt | Light microenvironment and single-cell gradients of carbon fixation in tissues of symbiont-bearing corals[END_REF]Cunning et al. 2015b). By applying the qPCR-HRM assay developed in this study we explored the abundance and repartitioning of Symbiodiniaceae clades at different spatial scales in A. pulchra. Our work revealed that the composition of Symbiodiniaceae clade assemblages in this coral species varied at all spatial scales investigated, including geographical (between two sites), inter-colony (within each site), and intra-colony (upward versus downward facing surface of the branch), confirming the high degree of spatial variation in endosymbiotic associations with Acropora species [START_REF] Ulstrup | Geographic and habitat partitioning of genetically distinct zooxanthellae (Symbiodinium) in Acropora corals on the Great Barrier Reef[END_REF][START_REF] Ulstrup | Inter-polyp genetic and physiological characterisation of Symbiodinium in an Acropora valida colony[END_REF][START_REF] Rouzé | An updated assessment of Symbiodinium spp. that associate with common scleractinian corals from Moorea (French Polynesia) reveals high diversity among background symbionts and a novel finding of clade B[END_REF].

At the geographical scale, significantly higher abundance of total Symbiodiniaceae were observed in A. pulchra originating from Mahana compared to Teavaro. The abundance of symbiont types can be enhanced by various environment factors, such as nutrient levels or turbidity [START_REF] Fabricius | Effects of terrestrial runoff on the ecology of corals and coral reefs: review and synthesis[END_REF]Cooper et al. 2011a). The higher total Symbiodiniaceae abundance at the Mahana site, which was characterized by higher turbidity corroborates the findings of [START_REF] Bay | Recovery from bleaching is mediated by threshold densities of background thermo-tolerant symbiont types in a reef-building coral[END_REF] that higher Symbiodiniaceae abundance in corals transplanted to inshore locations which are commonly associated with higher dissolved nutrients. As previously observed in Moorea [START_REF] Rouzé | An updated assessment of Symbiodinium spp. that associate with common scleractinian corals from Moorea (French Polynesia) reveals high diversity among background symbionts and a novel finding of clade B[END_REF], up to three distinct clades were detected in a single A. pulchra colony, which contrasts with findings of [START_REF] Thomas | Exploring Symbiodinium diversity and host specificity in Acropora corals from geographical extremes of Western Australia with 454 amplicon pyrosequencing[END_REF] who reported exclusive clade C dominance in A. pulchra colonies from Western Australia. In the present study, significant differences in the proportion of Symbiodiniaceae clades between sites were observed with a co-dominance of clades A and C in Teavaro compared to multi-clades A and D and clades A, C and D in Mahana. Despite the similar temperatures measured at both sites, (ranging from 28.7 to 28.9°C), a higher solar irradiance was recorded at Teavaro (average value: 1767 vs 2040 μmol s -1 m 2 ). Coral bleaching is often observed when high temperature and solar irradiance levels occur in combination [START_REF] Brown | Solar radiation modulates bleaching and damage protection in a shallow water coral[END_REF]. It has also been shown that, in addition to the symbiont identity, the abundance of Symbiodiniaceae cells within a coral may play a key role in influencing the function of coral-algal symbiosis, with clade C abundant corals being more prone to bleaching than those with abundant clade D or background C in branched corals [START_REF] Cunning | Excess algal symbionts increase the susceptibility of reef corals to bleaching[END_REF][START_REF] Cunning | Patterns of bleaching and recovery of Montipora capitata in Kāne'ohe Bay, Hawai'i, USA[END_REF]).

As such, the lower total Symbiodiniaceae density observed in the clade C dominated A. pulchra colonies from Teavaro (i.e., with higher light levels), could be the result of a mild bleaching event at that location, contrasting with the colonies at Mahana where the putative 'thermo-tolerant' clade D and 'opportunistic' clade A co-dominated and were present at higher densities.

Although the broad biological flexibility of Acropora sp. to interact with a wide range of Symbiodiniaceae genotypes may be advantageous under stable conditions (see [START_REF] Putnam | Endosymbiotic flexibility associates with environmental sensitivity in scleractinian corals[END_REF], the high incidence of clade A reported here in both studied sites may represent a lower resilience potential of coral species from Moorea lagoons. It has been suggested that a higher proportion of clade A in corals could increase their susceptibility to disease and Vibrio spp. colonization [START_REF] Rouzé | Symbiodinium clades A and D differentially predispose Acropora cytherea to disease and Vibrio spp. colonization[END_REF]. The omnipresence of clade A symbionts in A. pulchra from both sites is of concern for the health and future of corals in Moorea. Understanding the factors that trigger shifts in Symbiodiniaceae clades abundance and composition in corals around Moorea, through long-term analysis at different spatio-temporal scales, is an interesting avenue for future research.

Previous studies [START_REF] Ulstrup | Inter-polyp genetic and physiological characterisation of Symbiodinium in an Acropora valida colony[END_REF][START_REF] Kemp | A microsampling method for genotyping coral symbionts[END_REF][START_REF] Kemp | Community dynamics and physiology of Symbiodinium spp. before, during, and after a coral bleaching event[END_REF] [START_REF] Wangpraseurt | The in situ light microenvironment of corals[END_REF][START_REF] Wangpraseurt | Light microenvironment and single-cell gradients of carbon fixation in tissues of symbiont-bearing corals[END_REF], creating different niches at the micro-scale and favoring the establishment of different symbiotic clade partnerships that may help corals adapt to the prevailing conditions [START_REF] Kemp | A microsampling method for genotyping coral symbionts[END_REF][START_REF] Suggett | Functional diversity of photobiological traits within the genus Symbiodinium appears to be governed by the interaction of cell size with cladal designation[END_REF]. Our study reinforces earlier researches emphasizing the importance of intra-colony variation of Symbiodiniaceae and provides insights into the partitioning of Symbiodiniaceae clades within colonies of A. pulchra. Our work highlights the importance of considering micro-niches in studies linking symbiont diversity and coral response to environmental stress to assist in accurately predicting the impact of rising seawater temperature on coral populations and community responses.

In the present study, we developed and validated a qPCR-HRM assay. When coupled with clade and host specific qPCRs, it enabled the absolute quantification of endosymbiotic Symbiodiniaceae from all the nine clades from small coral samples, using the combination of family-Symbiodiniaceae and clades-specific primer sets. The method has the potential to provide useful data on the abundance and repartition of the endosymbiont clades at different biological scales (cells, polyp, colony, population and species), as well as different temporal and spatial scales, which may help understand the drivers behind symbiont community changes [START_REF] Chen | Fluctuating algal symbiont communities in Acropora palifera (Scleractinia: Acroporidae) from Taiwan[END_REF], or those following bleaching events [START_REF] Thornhill | Multi-year, seasonal genotypic surveys of coral-algal symbioses reveal prevalent stability or post-bleaching reversion[END_REF]. The method provides a new tool to describe the complexity of coral-Symbiodiniaceae symbiosis in a rapidly changing world. For example, it may contribute to elucidating the mechanisms governing symbiont shuffling as a result of frequent cumulative thermal stress and be useful to quantify horizontal and vertical transmission of symbionts from parental colonies to larvae, thus providing crucial information for restoration applications.

normalized HRM curves between clades presented in a). Dotted line represented the mean RMSE value obtained for pairwise comparisons within clades. . 2 Col. 3 Col. 4 Col. 5 Col. 6 Col. 7 Col. 8 Col. 1 Col. 2 Col. 3 Col. 4 Col. 5 Col. 6 Col. 7 Col. 8 Col. 9 Col. 10 Electronic Supplementary Material (Tables, Figures, Video, Movie, Audio, etc.)

  module of the LightCycler 480 v.1.5.0 Software (Roche Diagnostics, USA). Melting curve data was adjusted manually as previously described[START_REF] Meistertzheim | High Resolution Melting Analysis for fast and cheap polymorphism screening of marine populations[END_REF], and normalized fluorescence conditions were established and adjusted, with the threshold set to 0 and the sensitivity at 0.26[START_REF] Lavergne | Complex genetic structure of a euryhaline marine fish in temporarily open/closed estuaries from the wider Gulf of Aden[END_REF]. Curve shapes and Tm were compared between samples of the same clade and between clades. Shape similarities or differences in the melting curves between samples were assessed visually and mathematically by calculating the root-mean square error (RMSE) values. The latter were calculated by pairwise comparison of the normalized fluorescence data of melting curves obtained for each Symbiodiniaceae clade as recommended by Naue et al. (2014). The intra-and inter-run accuracy of HRM results were estimated by calculating the RMSE values of three runs of triplicate amplifications of all standard curves. The influence of template DNA was also investigated for each Symbiodiniaceae clade and embryos of A. pulchra at a DNA concentration range between 100 pg and 500 ng. Finally, to confirm the number of clades present in one sample by HRM, DNA from various clades was mixed in different proportions to obtain reference melting curves of DNA mixtures. DNA from clades A-D, F and G were cross-mixed in pairs using three different proportions of DNA (25:75, 50:50, 75:25) and by using three to five clades combined at equimolar proportion, and at a final DNA concentration of 15 ng DNA µL -1 .

(

  [START_REF] Bosserelle | Guide d'identification des coraux de Moorea[END_REF], twelve species representing eight different families were chosen, including Acroporidae (Acropora pulchra, Astreopora sp. and Montipora capitata), Agariciidae (Pachyseris speciosa and Pavona cactus), Faviidae (Dipsastraea [Favia] stelligera), Fungiidae (Danafungia sp.), Merulinidae (Cyphastrea serailia), Pocilloporidae (Pocillopora damicornis), Poritidae (Porites rus and Napopora irregularis) and Psammocoridae (Psammocora contigua).

  have described the differential distribution of in hospite Symbiodiniaceae clades at the micro-scale (i.e., within a colony), but without taking into account the absolute abundances of each clade and without measuring background symbiont genotypes. Interestingly, 22% of fragments collected from a branch (above vs below) of the same colony from Mahana harbored different proportions of Symbiodiniaceae. In this study, micro-scale variations revealed the presence of C-type populations in coral fragments originating from Mahana with lighter colors located below (shaded) compared to the ones located above branches. Although research efforts have focused on revealing the flexibility of coral symbiosis to changing environments and the existence of inter-colony variation among Symbiodiniaceae communities, comparatively little attention has been given to intra-colony variation. Rowan et al. (1997) emphasized intra-colony zonation of Symbiodiniaceae communities in response to environmental irradiance, suggesting micro-scale physiological acclimatization at the level of colony. Skeletal and tissue properties of corals alter in hospite light and oxygen availability to Symbiodiniaceae cells

Figure 4

 4 Figure 4Melting peaks using the 5.8S rRNA gene from mixtures observed in samples (solid line) and

Figure 5

 5 Figure 5 Cellular proportions of specific Symbiodiniaceae clades per total DNA (Symbiodiniaceae +

Figure 6

 6 Figure 6 Means proportions and standard deviations (SD) of specific Symbiodiniaceae clades per total

  Figure1

Table 1 .

 1 Combinations of forward and reverse primers used in real-time PCR and High Resolution Melting analyses.

	Longitude
	x
	y

Table 1 .

 1 Combinations of forward and reverse primers used in real-time PCR and High Resolution Melting analyses.

	Col. 11

Table 1

 1

Acknowledgements

This work was funded by the ANR « R-ECOLOGS » and the ANR JCJC « Live and let die ». Sincere thanks are due to Prof. Mary-Alice Coffroth from State University of New York at Buffalo (USA) and Dr. Hollie Putnam from University of Rhode Island (USA) for providing us with Symbiodiniaceae cultures and DNA samples, as well as Dr. Sarah Nahon from French National Institute for Agricultural Research (France) for providing pictures. The authors would like to thank the staff of the CRIOBE for their help during the experiments, and especially Antoine Puisay for his help in sampling corals.

Conflict of Interest:

The authors declare that they have no conflict of interest.

Ethical approval:

All applicable international, national, and/or institutional guidelines for the care and use of animals were followed. Coral collection was performed according to the French Polynesia regulation.

Author contribution

A.L.M., and L.H. designed research; A.L.M. performed research; A.L.M. and X.P. analyzed data; A.L.M. and X.P. wrote the paper; and J.F.G., S.A.W. and L.H. revised the paper.

Additional Information

The authors declare no conflict of interest. Electronic Supplementary Material (Tables ,Figures, Video, Movie, Audio, etc.) Supplemental data.pdf

Figure legend


Nicolas Danchin

Etienne Puymirat

Grégoire Lefèvre

Clément Delmas

Laurent Bonello

Patrick Henry

Eric Bonnefoy

Francois Schiele

Jean Ferrieres

Tabassome Simon

Nadia Aissaoui
email: nadia.aissaoui@aphp.fr

Elisabeth Riant

Jean Ferrières

The French registry of Acute ST-elevation and non-ST-elevation Myocardial Infarction (FAST-MI) programme

Keywords: AMI, acute myocardial infarction, CI, confidence interval, CS, cardiogenic shock, HR, hazard ratio, IABP, intra-aortic balloon pump, LV, left ventricular, LVEF, left ventricular ejection fraction, MI, myocardial infarction, NSTEMI, non-ST-segment elevation myocardial infarction, STEMI, ST-segment elevation myocardial infarction Acute myocardial infarction, Cardiogenic shock, Left ventricular function

ARTICLE IN PRESS

Résultats. -Parmi les 4169 patients ayant présenté un IDM, 3,3 % des patients ont présenté un CC dont 43 % avec FEVG > 40 %. L'angioplastie percutanée précoce (≤ 24 heures) était utilisée plus souvent chez les patients avec DVG (61 % vs 42 %). Le CC restait associé à une mortalité élevée à trois ans dans les deux groupes ; 55 % chez le groupe FEVG ≤ 40 % et 44 % dans le groupe FEVG > 40 %. En analyse multivariée, la DVG < 40 % était associée à une surmortalité à trois ans (HR 2,08, IC95 % 1,15-3,78) chez les patients en CC. L'analyse dans la cohorte 2005 retrouvait des résultats similaires. Conclusions. -Malgré une physiopathologie complexe du CC compliquant l'IDM, la DVG ≤ 40 % reste associé à une mortalité élevée à trois ans.

Background

Cardiogenic shock (CS) is the leading cause of hospital mortality associated with acute myocardial infarction (AMI) [START_REF] Califf | Cardiogenic shock[END_REF][START_REF] Topalian | Cardiogenic shock[END_REF]. Although the mortality associated with CS has decreased over the last 20 years, related to the broader use of percutaneous coronary intervention (PCI) and appropriate medications at the acute stage, it remains a major concern, with mortality rates reaching 45% at 1 month [START_REF] Aissaoui | Improved outcome of cardiogenic shock at the acute stage of myocardial infarction: a report from the USIK 1995, USIC 2000, and FAST-MI French nationwide registries[END_REF][START_REF] Berger | Impact of an aggressive invasive catheterization and revascularization strategy on mortality in patients with cardiogenic shock in the Global utilization of streptokinase and tissue plasminogen activator for occluded coronary arteries (GUSTO-I) trial. An observational study[END_REF][START_REF] Carnendran | Trends in cardiogenic shock: report from the SHOCK Study. The should we emergently revascularize occluded coronaries for cardiogenic shock?[END_REF][START_REF] Hochman | Cardiogenic shock complicating acute myocardial infarction-etiologies, management and outcome: a report from the SHOCK Trial Registry. Should we emergently revascularize occluded coronaries for cardiogenic shock?[END_REF][START_REF] Urban | A randomized evaluation of early revascularization to treat shock complicating acute myocardial infarction. The (Swiss) Multicenter Trial of Angioplasty for Shock-(S)MASH[END_REF].

Failure of the left ventricular (LV) pump is the primary insult in most forms of CS, but other parts of the circulatory system contribute to shock, with inadequate compensation or additional defects [START_REF] Reynolds | Cardiogenic shock: current concepts and improving outcomes[END_REF]. Indeed, the peripheral vasculature, neurohormonal and cytokine systems play a role in the pathogenesis and persistence of CS. In light of the complex pathophysiology of CS, LV ejection fraction (LVEF) may be only moderately depressed in CS complicating AMI without mechanical complications [START_REF] Reynolds | Cardiogenic shock: current concepts and improving outcomes[END_REF][START_REF] Chatterjee | Analytic reviews: cardiogenic shock with preserved systolic function: a reminder[END_REF]. In the SHould we emergently revascularize Occluded coronaries for Cardiogenic shocK (SHOCK) trial, mean LVEF was ≈ 30%. In the FAST-MI registries, we reported that 37% of patients with CS had LVEF > 40%, whereas 19% of patients without CS had LV dysfunction (< 40%) [START_REF] Hochman | Cardiogenic shock complicating acute myocardial infarction-etiologies, management and outcome: a report from the SHOCK Trial Registry. Should we emergently revascularize occluded coronaries for cardiogenic shock?[END_REF]. However, among patients with CS complicating AMI, previous studies have reported that LVEF remains a prognostic indicator [START_REF] Burns | The relationships of left ventricular ejection fraction, end-systolic volume index and infarct size to six-month mortality after hospital discharge following myocardial infarction treated by thrombolysis[END_REF][START_REF] Degeare | Predictive value of the Killip classification in patients undergoing primary percutaneous coronary intervention for acute myocardial infarction[END_REF][START_REF] Kernis | The incidence, predictors, and outcomes of early reinfarction after primary angioplasty for acute myocardial infarction[END_REF][START_REF] Marenzi | Impact of cardiac and renal dysfunction on inhospital morbidity and mortality of patients with acute myocardial infarction undergoing primary angioplasty[END_REF][START_REF] Moller | Prognostic importance of systolic and diastolic function after acute myocardial infarction[END_REF][START_REF] Picard | Echocardiographic predictors of survival and response to early revascularization in cardiogenic shock[END_REF][START_REF] Van Der Vleuten | The importance of left ventricular function for long-term outcome after primary percutaneous coronary intervention[END_REF][START_REF] Zaret | Value of radionuclide rest and exercise left ventricular ejection fraction in assessing survival of patients after thrombolytic therapy for acute myocardial infarction: results of thrombolysis in myocardial infarction (TIMI) phase II study[END_REF]. Overall in patients with CS, little is known about the clinical presentation, management and long-term mortality according to early LV function (i.e. assessed at the acute phase of AMI).

The aim of the present study was to assess presentation, management and clinical outcomes in patients admitted for AMI with CS, according to early LVEF, using long-term data from the French registry of Acute ST-elevation or non-STelevation Myocardial Infarction (FAST-MI) 2010.

Methods

FAST-MI 2010 registry

FAST-MI 2010 is a national prospective multicentre registry including consecutive adult patients hospitalized for ST-segment elevation and non-ST-segment elevation AMI (STEMI and NSTEMI) (with symptom onset ≤ 48 hours) over a period of 1 month (from October 2010). Patients with AMI after cardiovascular procedures were excluded. Participation in the study was offered to all intensive care units in French institutions (university teaching hospitals, general and regional hospitals and private clinics) with the capacity to receive acute coronary syndrome emergencies. Details of the methodology have been described previously [START_REF] Cambou | The French registry of acute ST elevation or non-ST-elevation myocardial infarction (FAST-MI): study design and baseline characteristics[END_REF][START_REF] Hanssen | French Registry on acute ST-elevation and non ST-elevation myocardial infarction 2010. FAST-MI 2010[END_REF]. The main objective of this registry was to evaluate strategies for the management of myocardial infarction (MI) in routine practice and to measure their association with outcomes over a 10-year follow-up. The registry was conducted in compliance with Good Clinical Practice guidelines, French law and the French data protection law. The protocol was reviewed and approved by the Committee for the Protection of Human Subjects of Saint-Louis University Hospital, and the FAST-MI data file was declared to the Commission Nationale Informatique et Liberté. The trial is registered at Clinicaltrials.gov: identifier NCT01237418.

Study population

A total of 4169 patients in 213 centres (76% of active centres in France) were included in this registry. Baseline characteristics were collected prospectively. All data were recorded on computerized case record forms by dedicated research technicians sent to each of the centres at least once a week. The research technicians were also asked to ensure that recruitment was consecutive. In the current analysis, we selected all patients with AMI (STEMI and NSTEMI) with CS.

CS was defined as systolic blood pressure < 90 mmHg in the absence of hypovolaemia, and associated with cyanosis, cold extremities, changes in mental status, persistent oliguria or congestive heart failure. Decreased LVEF was defined as LVEF ≤ 40% [START_REF] Ponikowski | ESC Guidelines for the diagnosis and treatment of acute and chronic heart failure: The Task Force for the diagnosis and treatment of acute and chronic heart failure of the European Society of Cardiology (ESC). Developed with the special contribution of the Heart Failure Association (HFA) of the ESC[END_REF]. The last LVEF assessed during the stay in the intensive care unit was analysed.

Statistical analysis

Statistical analysis was performed using SPSS premium statistics software, version 23.0 (IBM, Armonk, NY, USA). For quantitative variables, means and standard deviations were calculated. In addition, medians with interquartile ranges were calculated when appropriate. Discrete variables are presented as numbers of events and percentages. Comparisons were made with the 2 test or Fisher's exact tests for discrete variables, and by unpaired t tests, Mann-Whitney tests or one-way analyses of variance for continuous variables. Survival curves were estimated using Kaplan-Meier estimators, and were compared using log-rank tests. Correlates of survival were determined using a multivariable backward stepwise Cox analysis. Cumulative hazard functions were computed to assess proportionality. The candidate variables included in the multivariable analyses were selected ad hoc, based upon their physiological relevance and potential to be associated with long-term mortality. To adjust for confounders, in addition to LV function, we adjusted for age, sex, type of AMI, risk factors, non-cardiovascular co-morbidity, medications and revascularization (variables listed in Table 1 andTable 2) in the whole population. For all analyses, a P value < 0.05 was considered significant.

For assessing the robustness of the results, we repeated the analysis in patients with CS in the FAST-MI 2005 registry, which had been carried out five years earlier, using similar methodology (Clinicaltrials.gov identifier: NCT00673036) [START_REF] Cambou | The French registry of acute ST elevation or non-ST-elevation myocardial infarction (FAST-MI): study design and baseline characteristics[END_REF]. The set of variables collected in 2005 was essentially similar to that collected in 2010.

Results

Main analysis: FAST-MI 2010

Characteristics and clinical presentation

Of the 4169 patients included in the FAST-MI 2010 registry, 138 (3.3%) patients with AMI developed CS during hospitalization, and 112 had LVEF data available (Table 1). Fifty-seven percent of patients with CS had LV systolic dysfunction. The two groups (LVEF ≤ 40% and LVEF > 40%) did not differ in many respects, particularly regarding past medical history. However, patients with decreased LVEF were significantly younger (70.1 ± 12.9 vs 75.5 ± 12.3 years). The GRACE (Global Registry of Acute Coronary Events) score was similar in both groups (201 ± 42 vs 204 ± 37). Clinical presentation at admission was slightly different according to LV function, with numerically higher proportions of STEMI, cardiac arrest and typical chest pain in patients with LV systolic dysfunction. The left anterior descending artery was the most frequent culprit lesion in patients with LVEF ≤ 40%, while the right coronary artery was the most frequent culprit lesion in patients with LVEF > 40%. Finally, multivessel coronary artery disease was slightly more frequent in patients with LVEF ≤ 40% (Table 2).

Initial management

Initial management is presented in Table 2. Numerically more patients with decreased LVEF received prasugrel, glycoprotein IIb/IIIa inhibitors and unfractionated heparin. The use of life-support therapies (inotropes and intra-aortic balloon pump [IABP]) were significantly higher in patients with LV systolic dysfunction, but < 50% of the patients with CS received inotropes in the first 48 hours after admission.

In patients with STEMI, the rate of primary PCI was numerically higher in patients with LVEF ≤ 40%, and the time from electrocardiogram to primary PCI was significantly higher in these patients (median [IQR]: 127 [95; 258] vs 116 [78; 191] minutes). In patients with NSTEMI, the rate of early PCI (i.e. performed during the first 24 hours) was numerically higher in patients with decreased LVEF.

In-hospital complications and clinical outcomes

In-hospital complications (recurrent MI, intrastent thrombosis, atrial fibrillation, stroke and bleeding) were similar regardless of LVEF. The use of blood transfusion was also similar in both groups. In-hospital death was numerically higher in patients with LVEF ≤ 40% (36% vs 29%), as was mortality at 1 year (47% vs 35%) and 3 years (55% vs 44%) (Table 3).

Using multivariable analysis, early LV systolic dysfunction was associated with a higher 3-year mortality risk (hazard ratio [HR] 2.08; 95% confidence interval [CI] 1.15-3.78) (Fig. 1 andTable A.1]. In patients with NSTEMI, LVEF ≤ 40% was associated with a high risk of mortality at 3 years after adjustment (HR 5.29, 95% CI 1.71-16.44), while only

Replication cohort: FAST-MI 2005

Among the 3670 patients included in the FAST-MI 2005 registry, 224 (6.1%) patients with AMI developed CS during hospitalization (164 patients with LVEF data available). Baseline characteristics did not differ between patients with CS with or without LV systolic dysfunction (Table A.2 and Table A.3). In-hospital complications (recurrent MI, intrastent thrombosis, atrial fibrillation, stroke and bleeding) were also similar regardless of the LVEF (Table A.4). In-hospital death was numerically higher in patients with LVEF ≤ 40% (50% vs 41%), as was mortality at 1 year (67% vs 49%) and three years (75% vs 52%). Using multivariable analysis, LV systolic dysfunction was associated with higher 3-year mortality in the overall population (HR 1.96, 95% CI 1.29-3.00), as well as in patients with STEMI (HR 1.94, 95% CI 1.11-3.38) and patients with NSTEMI (HR 2.68, 95% CI 1.30-5.12) (Table A.5).

Discussion

The present data from a nationwide registry indicate that approximately 40% of patients who develop CS during AMI have no severe LV systolic dysfunction. Among patients with CS, there was only little difference between those with or without LV systolic dysfunction; from an anatomical standpoint, CS was more often related to left anterior descending artery involvement in those with low LVEF, while the right coronary artery was the predominant culprit artery in those without severe LV systolic dysfunction. The management of the two LVEF groups did not differ essentially, except for more frequent use of life-support therapies (inotropes and IABP) in those with low LVEF. LVEF ≤ 40%, however, was associated with higher long-term mortality after full adjustment for clinical presentation and management. LV dysfunction is an established correlate of increased short-and long-term mortality after AMI [START_REF] Burns | The relationships of left ventricular ejection fraction, end-systolic volume index and infarct size to six-month mortality after hospital discharge following myocardial infarction treated by thrombolysis[END_REF][START_REF] Degeare | Predictive value of the Killip classification in patients undergoing primary percutaneous coronary intervention for acute myocardial infarction[END_REF][START_REF] Kernis | The incidence, predictors, and outcomes of early reinfarction after primary angioplasty for acute myocardial infarction[END_REF][START_REF] Marenzi | Impact of cardiac and renal dysfunction on inhospital morbidity and mortality of patients with acute myocardial infarction undergoing primary angioplasty[END_REF][START_REF] Moller | Prognostic importance of systolic and diastolic function after acute myocardial infarction[END_REF][START_REF] Van Der Vleuten | The importance of left ventricular function for long-term outcome after primary percutaneous coronary intervention[END_REF][START_REF] Zaret | Value of radionuclide rest and exercise left ventricular ejection fraction in assessing survival of patients after thrombolytic therapy for acute myocardial infarction: results of thrombolysis in myocardial infarction (TIMI) phase II study[END_REF]. Several studies have demonstrated that patients with STEMI with LV dysfunction have increased rates of mortality, major adverse cardiovascular events (MACE) and major bleeding [START_REF] Halkin | Impact and determinants of left ventricular function in patients undergoing primary percutaneous coronary intervention in acute myocardial infarction[END_REF][START_REF] Ng | The prognostic importance of left ventricular function in patients with ST-segment elevation myocardial infarction: the HORIZONS-AMI trial[END_REF]. However, in most reports examining the significance of LVEF after MI, LVEF assessment occurred later during the healing process, and very few data are available related to LVEF and dysfunction in patients with AMI with CS. Our data show that early LV systolic dysfunction affects 57% of patients with AMI with CS. Since the SHOCK study, the complex pathophysiology of CS has been highlighted, and it is now recognized that LVEF may be only moderately depressed in CS complicating AMI without mechanical complications [START_REF] Reynolds | Cardiogenic shock: current concepts and improving outcomes[END_REF][START_REF] Chatterjee | Analytic reviews: cardiogenic shock with preserved systolic function: a reminder[END_REF]. However, a rate of 40% of patients with CS having an LVEF > 40% may appear high. The assessment of cardiac function is difficult, and LVEF has many determinants: not only cardiac performance, but also preload, afterload, heart rate and synchronization [START_REF] Carabello | Evolution of the study of left ventricular function: everything old is new again[END_REF]. Of note, patients with LVEF > 40% were older compared with those with decreased LVEF. Subanalyses according to type of MI suggest that the proportion of patients with CS with LVEF > 40% was increased in NSTEMI compared with STEMI, which can be explained by the clinical characteristics of both populations. Typically, patients with NSTEMI are older and have more frequent risk factors (especially hypertension), co-morbidities and a higher burden of coronary artery disease [START_REF] Chan | Long-term mortality of patients undergoing cardiac catheterization for ST-elevation and non-ST-elevation myocardial infarction[END_REF][START_REF] Hasdai | A prospective survey of the characteristics, treatments and outcomes of patients with acute coronary syndromes in Europe and the Mediterranean basin; the Euro Heart Survey of Acute Coronary Syndromes (Euro Heart Survey ACS)[END_REF][START_REF] Steg | Baseline characteristics, management practices, and in-hospital outcomes of patients hospitalized with acute coronary syndromes in the Global Registry of Acute Coronary Events (GRACE)[END_REF]. These characteristics could contribute to LV diastolic dysfunction. Indeed, it is likely that abnormalities of ventricular relaxation and compliance contribute to CS in some cases [START_REF] Reynolds | Cardiogenic shock: current concepts and improving outcomes[END_REF].

Interestingly, the use of inotrope therapy during the first 48 hours after admission was increased in patients with LV dysfunction, but remained < 50% in both groups. This finding may arise from the fact that CS might have developed at a later stage after admission, from a lack of evidence for clinical benefits of inotropes and from the fear of potential harm with inotropes in CS, resulting in disparities in clinical practice [START_REF] Ponikowski | ESC Guidelines for the diagnosis and treatment of acute and chronic heart failure: The Task Force for the diagnosis and treatment of acute and chronic heart failure of the European Society of Cardiology (ESC). Developed with the special contribution of the Heart Failure Association (HFA) of the ESC[END_REF][START_REF] Levy | Experts' recommendations for the management of adult patients with cardiogenic shock[END_REF]. Finally, increased use of circulatory assist devices may explain the reduced catecholamine requirements [START_REF] Ponikowski | ESC Guidelines for the diagnosis and treatment of acute and chronic heart failure: The Task Force for the diagnosis and treatment of acute and chronic heart failure of the European Society of Cardiology (ESC). Developed with the special contribution of the Heart Failure Association (HFA) of the ESC[END_REF][START_REF] Levy | Experts' recommendations for the management of adult patients with cardiogenic shock[END_REF]. In our population, the use of assist devices and IABP was also increased in patients with LVEF < 40%. IABP has been used widely as mechanical support in CS [START_REF] Kantrowitz | Initial clinical experience with intraaortic balloon pumping in cardiogenic shock[END_REF]. However, the IABP-SHOCK II trial, including 600 patients with AMI with CS, did not show any beneficial effect on 30-day mortality with the use of IABP [START_REF] Thiele | Intra-aortic balloon counterpulsation in acute myocardial infarction complicated by cardiogenic shock (IABP-SHOCK II): final 12 month results of a randomised, open-label trial[END_REF][START_REF] Thiele | Intraaortic balloon support for myocardial infarction with cardiogenic shock[END_REF]. Thus, the use of IABP for this indication is not routinely recommended [START_REF] Ponikowski | ESC Guidelines for the diagnosis and treatment of acute and chronic heart failure: The Task Force for the diagnosis and treatment of acute and chronic heart failure of the European Society of Cardiology (ESC). Developed with the special contribution of the Heart Failure Association (HFA) of the ESC[END_REF][START_REF] Thiele | Intra-aortic balloon counterpulsation in acute myocardial infarction complicated by cardiogenic shock (IABP-SHOCK II): final 12 month results of a randomised, open-label trial[END_REF][START_REF] Windecker | ESC/EACTS guidelines on myocardial revascularization: the Task Force on Myocardial Revascularization of the European Society of Cardiology (ESC) and the European Association for Cardio-Thoracic Surgery (EACTS). Developed with the special contribution of the European Association of Percutaneous Cardiovascular Interventions (EAPCI)[END_REF]. The LVEF assessments were not performed at the same time of hospital stay, and could be done with or without inotropic and IABP support. However, the in SHOCK trial, LVEF was usually measured while patients were on inotropic and/or balloon support, and the authors reported that LVEF was similar in the acute phase of CS and two weeks later, when functional status was quite different [START_REF] Reynolds | Cardiogenic shock: current concepts and improving outcomes[END_REF][START_REF] Picard | Echocardiographic predictors of survival and response to early revascularization in cardiogenic shock[END_REF].

Finally, LVEF ≤ 40% at the acute phase of AMI with CS was associated with higher long-term mortality after full adjustment for clinical presentation and management. These findings were consistent with previous studies [START_REF] Burns | The relationships of left ventricular ejection fraction, end-systolic volume index and infarct size to six-month mortality after hospital discharge following myocardial infarction treated by thrombolysis[END_REF][START_REF] Degeare | Predictive value of the Killip classification in patients undergoing primary percutaneous coronary intervention for acute myocardial infarction[END_REF][START_REF] Kernis | The incidence, predictors, and outcomes of early reinfarction after primary angioplasty for acute myocardial infarction[END_REF][START_REF] Marenzi | Impact of cardiac and renal dysfunction on inhospital morbidity and mortality of patients with acute myocardial infarction undergoing primary angioplasty[END_REF][START_REF] Moller | Prognostic importance of systolic and diastolic function after acute myocardial infarction[END_REF][START_REF] Picard | Echocardiographic predictors of survival and response to early revascularization in cardiogenic shock[END_REF][START_REF] Van Der Vleuten | The importance of left ventricular function for long-term outcome after primary percutaneous coronary intervention[END_REF][START_REF] Zaret | Value of radionuclide rest and exercise left ventricular ejection fraction in assessing survival of patients after thrombolytic therapy for acute myocardial infarction: results of thrombolysis in myocardial infarction (TIMI) phase II study[END_REF], and may support a strategy of aggressive manoeuvres to enhance forward LV stroke volume and improve regional and global LV systolic function in patients with CS caused by extensive LV dysfunction [START_REF] Reynolds | Cardiogenic shock: current concepts and improving outcomes[END_REF].

Study limitations

The study suffers from the same limitation as all observational studies, i.e. no causality can be asserted between variables that are correlated. Comparisons between patients according to LV systolic function were not randomized and, despite careful adjustments for a large number of potentially confounding variables, and the use of statistical adjustment with the inverse probability weighting, the results can only be considered indicative. Only medications administered during the first 48 hours and/or at discharge were recorded. Finally, LVEF was not available in a number of patients.

Conclusions

In conclusion, CS carries a very high short-term and long-term mortality risk, irrespective of initial LV systolic dysfunction. Low LVEF in patients with CS, however, has an independent prognostic significance regarding long-term mortality.

Role of the funding source

The FAST-MI 2010 is a registry of the French Society of Cardiology, supported by unrestricted grants from Merck, the Eli-Lilly-Daiichi-Sankyo alliance, AstraZeneca, Sanofi-Aventis, GSK and Novartis. FAST-MI 2005 is a registry of the French Society of Cardiology, supported by unrestricted grants from Pfizer and Servier. Additional support was obtained from a research grant from the French Caisse Nationale d'assurance maladie.

Figure 1 .

 1 Figure 1. Long-term survival in patients with cardiogenic shock, according to left ventricular function (FAST-MI 2010 cohort).

Table 1

 1 Baseline characteristics and clinical presentation in patients with cardiogenic shock, according to left ventricular function (FAST-MI 2010 cohort).

		LV function ≤ 40%(n = 64)	LV function > 40%(n = 48)	P
	Age (years)	70.1 ± 12.9	75.5 ± 12.3	0.03
	Age > 75 years	27 (42)	30 (62.5)	0.03
	Body mass index (kg/m 2 )	26.7 ± 4.7	27.3 ± 5.8	0.56
	Women	17 (27)	31 (65)	0.19
	Risk factors
	Hypertension	44 (69)	35 (73)	0.63
	Diabetes	42 (34)	15 (31)	0.73
	Dyslipidaemia	30 (47)	26 (54)	0.45
	Current smoker	12 (19)	41 (15)	0.56
	Cardiovascular history and co-morbidities
	History of MI	12 (19)	8 (17)	0.78
	Previous PCI	10 (16)	6 (12.5)	0.64
	Previous CABG	5 (8)	1 (2)	0.18
	History of heart failure	6 (9)	6 (12.5)	0.60
	History of stroke	2 (3)	0 (0)	0.22
	Peripheral artery disease	8 (12.5)	6 (12.5)	1.0
	Chronic renal failure	6 (9)	3 (6)	0.55
	Medication history
	Aspirin	15 (23)	14 (29)	0.67
	Clopidogrel	9 (14)	4 (31)	0.35
	Beta-blocker	19 (30)	21 (44)	0.12
	Statin	19 (30)	21 (44)	0.12
	ACE inhibitor or ARB	20 (31)	28 (58)	0.004
	Clinical presentation
	STEMI	19 (70)	28 (58)	0.19
	Typical chest pain	52 (81)	26 (54)	0.002
	Cardiac arrest	5 (8)	2 (4)	0.43
	GRACE score	201 ± 42	204 ± 37	0.78

Data are expressed as mean ± standard deviation or number (%). ACE: angiotensin-converting enzyme; ARB: angiotensin receptor blocker; CABG: coronary artery bypass graft; GRACE: Global Registry of Acute Coronary Events; LV: left ventricular; MI: myocardial infarction; PCI: percutaneous coronary intervention; STEMI: ST-segment elevation myocardial infarction.

Table 2

 2 In-hospital management of patients with cardiogenic shock, according to left ventricular function (FAST-MI 2010 cohort).

		LV function ≤ 40%(n = 64)	LV function > 40%(n = 48)	P
	Aspirin	57 (89)	46 (96)	0.19
	Clopidogrel	44 (69)	41 (85)	0.04
	Prasugrel	10 (16)	6 (12.5)	0.64
	GP IIb/IIIa	25 (39)	12 (25)	0.18
	UFH	42 (66)	27 (56)	0.31
	LMWH	23 (36)	21 (44)	0.40
	Bivalirudine	5 (8)	2 (4)	0.43
	Fondaparinux	6 (9)	6 (12.5)	0.60
	Proton pump inhibitor	35 (55)	30 (62.5)	0.41
	Statin	46 (72)	35 (73)	0.90
	Beta-blocker	23 (36)	21 (44)	0.40
	ACE inhibitor	26 (41)	16 (33)	0.43
	Inotrope	30 (47)	10 (19)	0.002
	Diuretic	40 (62.5)	28 (58)	0.66
	Femoral access	24 (56)	20 (44)	0.24
	Drug-eluting stent	13 (27)	8 (23)	0.67
	IABP	25 (39)	8 (17)	0.01
	Other short-term assist devices	6 (9.5)	2 (4)	0.29
	Management in patients with STEMI
	ECG to primary PCI (minutes)	127 [95; 258] (n = 31)	116 [78; 191] (n = 16)	< 0.001
	Primary PCI	33 (77)	16 (59)	0.12
	Thromboaspiration	14 (37)	10 (48)	0.72
	Fibrinolysis	3 (7)	3 (11)	0.54
	Management in patients with NSTEMI
	PCI ≤ 24 hours	6 (32)	4 (20)	0.11
	PCI during hospitalization	10 (53)	14 (70)	0.27
	CAG during hospitalization	15 (79)	16 (80)	0.94
	Culprit lesion
	Left main coronary artery	4 (6)	2 (4)	0.26
	Left anterior descending artery	26 (41)	9 (19)
	Circumflex artery	8 (12.5)	8 (17)
	Right coronary artery	15 (23)	20 (42)

Data are expressed as number (%) or median [interquartile range]. ACE: angiotensin-converting enzyme; CAG: coronary angiography; ECG: electrocardiogram; GP: glycoprotein; IABP: intra-aortic balloon pump; LMWH: low-molecular-weight heparin; LV: left ventricular; NSTEMI: non-ST-segment elevation myocardial infarction; PCI: percutaneous coronary intervention; STEMI: ST-segment elevation myocardial infarction; UFH: unfractionated heparin.

Table 3

 3 In-hospital complications and clinical outcomes in patients with cardiogenic shock, according to left ventricular function (FAST-MI 2010 cohort).

		LV function ≤ 40%(n = 64)	LV function > 40%(n = 48)	P
	Recurrent MI	2 (3)	1 (2)	0.74
	Intrastent thrombosis	2 (3)	1 (2)	0.74
	Atrial fibrillation	14 (22)	7 (15)	0.33
	New AV block	10 (16)	6 (12.5)	0.59
	Ventricular fibrillation	9 (14)	3 (6)	0.19
	Ventricular tachycardia	9 (14)	3 (6)	0.19
	Stroke	1 (2)	0 (0)	0.38
	Major bleeding	3 (5)	3 (6)	0.71
	Minor bleeding	3 (5)	3 (6)	0.71
	Red cell transfusions	9 (14)	5 (10)	0.56
	In-hospital death	23 (36)	14 (29)	0.29
	Death at 1 year	30 (47)	17 (35)	0.29
	Death at 3 years	35 (55)	21 (44)	0.13

Data are expressed as number (%). AV: atrioventricular; LV: left ventricular; MI: myocardial infarction.

Acknowledgements

The authors are deeply indebted to the patients who agreed to participate and to all the physicians who took care of them. Special thanks to all involved in the collection and analysis of the data: ICTA contract research organization (Fontaine-lès-Dijon, France) and the devoted personnel of URCEST (Assistance Publique des hôpitaux de Paris and University Paris 6) and INSERM U 1027 (Toulouse). Special thanks to Vincent Bataille, PhD, for his careful data management, to Benoît Pace (Société franc ¸aise de cardiologie) for his invaluable assistance in designing the electronic case record form and to Geneviève Mulak, Pharm D (Société franc ¸aise de cardiologie) and Élodie Drouet, MSc, who supervised patient follow-up.

Disclosure of interest

The authors declare that they have no competing interest.

Appendix A. Supplementary data

Supplementary data associated with this article can be found, in the online version, at https://doi.org/10.1016/j.acvd.2017.11.002.