H J Alves Monteiro

C Brahmi

A B Mayfield

J Vidal-Dupiol

B Lapeyre

Jeremy Le Luyer
email: jeremy.le.luyer@ifremer.fr

Alves Monteiro

Le Luyer

Molecular mechanisms of acclimation to long-term elevated temperature exposure in marine symbioses

Keywords: co-expression network analysis, giant clams, metabarcoding, RNA-Seq, Symbiodiniaceae, thermoacclimation

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Molecular mechanisms of acclimation to long-term elevated temperature exposure in marine symbioses

Introduction

The "small giant" clams (Tridacna maxima; hereafter referred to as simply "clams") are mixotrophic organisms living in obligatory symbiosis with photosynthetic dinoflagellates of the family Symbiodiniaceae [START_REF] Holt | Photosymbiotic giant clams are transformers of solar flux[END_REF][START_REF] Jantzen | Photosynthetic performance of giant clams, Tridacna maxima and T. squamosa, Red Sea[END_REF][START_REF] Lajeunesse | Systematic Revision of Symbiodiniaceae Highlights the Antiquity and Diversity of Coral Endosymbionts[END_REF]. Symbiodiniaceae associate not only with clams, but with a diverse array of marine invertebrates, namely sponges, molluscs, and cnidarians; indeed, the coral-Symbiodiniaceae symbiosis is the functional basis of all coral reefs [START_REF] Hughes | Climate change, human impacts, and the resilience of coral reefs[END_REF]. Whereas in scleractinian corals symbionts are located intracellularly, in clams they reside extracellularly inside a tubular system ("Z-tubules"), which is 1) found in the outer epithelium of the mantle and 2) connected to the stomach [START_REF] Norton | The zooxanthellal tubular system in the Giant clam[END_REF]. These in hospite dinoflagellates are known to provide nutrients to their clam hosts via photosynthesis and may account for a major part of the clams' energy needs (depending on the species and the life history stage) [START_REF] Hawkins | Nutrition of the giant clam Tridacna gigas (L.). II. Relative contributions of filter-feeding and the ammonium-nitrogen acquired and recycled by symbiotic alga towards total nitrogen requirements for tissue growth and metabolism[END_REF][START_REF] Klumpp | Contributions of phototrophic and heterotrophic nutrition to the metabolic and growth requirements of four species of giant clam (Tridacnidae)[END_REF][START_REF] Klumpp | Contributions of phototrophic and heterotrophic nutrition to the metabolic and growth requirements of four species of giant clam (Tridacnidae)[END_REF][START_REF] Lucas | The biology, exploitation, and mariculture of giant clams (Tridacnidae)[END_REF][START_REF] Soo | The behaviour of giant clams (Bivalvia: Cardiidae: Tridacninae)[END_REF].

The systematics of the family Symbiodiniaceae have recently been revised to include at least nine different genera (formerly referred to as "clades") with well characterized molecular and physiological differences [START_REF] Lajeunesse | Systematic Revision of Symbiodiniaceae Highlights the Antiquity and Diversity of Coral Endosymbionts[END_REF]. One Symbiodiniaceae genus, formerly known as clade A (which includes the species Symbiodinium fitti, S. microadriaticum, and S. tridacnidorum), has been recurrently found in symbiosis with T. maxima, though members of clades C (Cladocopium) and D (Durusdinium) have been found in clam tissues, as well [START_REF] Baillie | Conspecificity and Indo-Pacific distribution of Symbiodinium genotypes (Dinophyceae) from giant clams[END_REF][START_REF] Deboer | Patterns of Symbiodinium distribution in three giant clam species across the biodiverse Bird's Head region of Indonesia[END_REF][START_REF] Ikeda | Zooxanthellal genetic varieties in giant clams are partially determined by species-intrinsic and growth-related characteristics[END_REF][START_REF] Lajeunesse | Investigating the biodiversity, ecology, and phylogeny of endosymbiotic dinoflagellates in the genus Symbiodinium using the ITS region: in search of a "species" level marker[END_REF][START_REF] Lee | Symbiodinium tridacnidorum sp. nov., a dinoflagellate common to Indo-Pacific giant clams, and a revised morphological description of Symbiodinium microadriaticum Freudenthal, emended Trench Blank[END_REF][START_REF] Mies | Molecular evidence of symbiotic activity between Symbiodinium and Tridacna maxima larvae[END_REF][START_REF] Pinzón | Microsatellite loci for Symbiodinium A3 (S. fitti) a common algal symbiont among Caribbean Acropora (stony corals) and Indo-Pacific giant clams (Tridacna)[END_REF].

Depending on the clam species, the symbiont assemblage has been found to vary with individual size (mostly observed in T. squamosa), as well as across environmental gradients (especially seawater temperatures) [START_REF] Deboer | Patterns of Symbiodinium distribution in three giant clam species across the biodiverse Bird's Head region of Indonesia[END_REF][START_REF] Ikeda | Zooxanthellal genetic varieties in giant clams are partially determined by species-intrinsic and growth-related characteristics[END_REF].

In French Polynesia, eastern Tuamotu's archipelagos were historically characterized by high densities of clams [START_REF] Andréfouët | Climate variability and massive mortalities challenge giant clam conservation and management efforts in French Polynesia atolls[END_REF][START_REF] Gilbert | Extraordinarily high giant clam density under protection in Tatakoto atoll (Eastern Tuamotu archipelago, French Polynesia)[END_REF][START_REF] Gilbert | Special traits and promises of the giant clam (Tridacna maxima) in French Polynesia[END_REF]. Recent mortality episodes and/or "bleaching" events in the Tuamotu Islands have, however, been reported, including 1) a massive mortality event in 2009 that reduced the clam population by 90% at Tatakoto Atoll [START_REF] Andréfouët | Climate variability and massive mortalities challenge giant clam conservation and management efforts in French Polynesia atolls[END_REF]Van Wynsberge, Andréfouët, GaertnerMazouni, & Remoissenet, 2018) and 2) a bleaching event in 2016 that affected 77 and 90% of the wild and cultured giant clam populations, respectively, at Reao Atoll [START_REF] Andréfouët | Adaptive management for the sustainable exploitation of lagoon resources in remote islands: lessons from a massive El Niño-induced giant clam bleaching event in the Tuamotu atolls (French Polynesia)[END_REF].

An increase in surface seawater temperature over a prolonged period (approximately three months above 30°C) is suspected to have triggered such bleaching events [START_REF] Andréfouët | Climate variability and massive mortalities challenge giant clam conservation and management efforts in French Polynesia atolls[END_REF][START_REF] Andréfouët | Adaptive management for the sustainable exploitation of lagoon resources in remote islands: lessons from a massive El Niño-induced giant clam bleaching event in the Tuamotu atolls (French Polynesia)[END_REF][START_REF] Van Wynsberge | Consequences of an uncertain mass mortality regime triggered by climate variability on giant clam population management in the Pacific Ocean[END_REF].

As with corals, bleaching in clams corresponds to the loss of symbiotic Symbiodiniaceae from the hosts [START_REF] Andréfouët | Climate variability and massive mortalities challenge giant clam conservation and management efforts in French Polynesia atolls[END_REF][START_REF] Buck | Effect of increased irradiance and thermal stress on the symbiosis of Symbiodinium microadriaticum and Tridacna gigas[END_REF][START_REF] Fitt | Coral bleaching: interpretation of thermal tolerance limits and thermal thresholds in tropical corals[END_REF]HoeghGuldberg et al., 2007;[START_REF] Leggat | The impact of bleaching on the metabolic contribution of dinoflagellate symbionts to their giant clam host[END_REF]. Symbiodiniaceae community variability and diversity (i.e., the collective assemblage of various genera and/or species) seems to be a determining factor in the sensitivity and resilience of both coral and clam hosts to increased temperatures [START_REF] Barshis | Lineage-Specific Transcriptional Profiles of Symbiodinium spp. Unaltered by Heat Stress in a Coral Host[END_REF][START_REF] Barshis | Genomic basis for coral resilience to climate change[END_REF][START_REF] Ladner | Protein evolution in two co-occurring types of Symbiodinium: an exploration into the genetic basis of thermal tolerance in Symbiodinium clade D[END_REF][START_REF] Rowan | Landscape ecology of algal symbionts creates variation in episodes of coral bleaching[END_REF]. However, the cell physiology of the host and symbionts is likely to be as important, if not more so, than the Symbiodiniaceae assemblage, in terms of gauging the ability of the clam-Symbiodiniaceae symbiosis to acclimate to elevated temperature over prolonged durations.

To date, few studies have investigated the transcriptomic response of giant clams to elevated temperatures; lipid profiling analyses are more routinely undertaken [START_REF] Dubousquet | Changes in fatty acid composition in the giant clam Tridacna maxima in response to thermal stress[END_REF]. The transcriptomic response to elevated temperature of several other taxa, mostly scleractinian coral species [START_REF] Crowder | Impacts of temperature and lunar day on gene expression profiles during a monthly reproductive cycle in the brooding coral Pocillopora damicornis[END_REF][START_REF] Hou | RNA-Seq reveals extensive transcriptional response to heat stress in the stony coral Galaxea fascicularis[END_REF][START_REF] Kenkel | Gene expression plasticity as a mechanism of coral adaptation to a variable environment[END_REF][START_REF] Pinzón | Whole transcriptome analysis reveals changes in expression of immune-related genes during and after bleaching in a reefbuilding coral[END_REF] and cultured Symbiodiniaceae [START_REF] Gierz | Transcriptomic analysis of thermally stressed Symbiodinium reveals differential expression of stress and metabolism genes[END_REF][START_REF] Levin | Sex, scavengers, and chaperones: transcriptome secrets of divergent Symbiodinium thermal tolerances[END_REF] have also been explored, yet few studies have looked at the mRNA level responses of multiple Symbiodiniaceae clades and host systems in the same study. Furthermore, few physiological data and even fewer transcriptomic data are available for the high-temperature responses of the giant clam T. maxima and its symbionts [but see [START_REF] Dubousquet | Changes in fatty acid composition in the giant clam Tridacna maxima in response to thermal stress[END_REF][START_REF] Zhou | Oxidative stress, apoptosis activation and symbiosis disruption in giant clam Tridacna crocea under high temperature[END_REF]]; these two published studies, though, only considered the response to an abrupt, rapid increase in temperature (short-term stress response).

Consequently, our understanding of the possible key drivers in high-temperature acclimation remains largely incomplete, despite its importance in generating better predictions of the impact of climate change on wild populations of giant clams [START_REF] Van Wynsberge | Consequences of an uncertain mass mortality regime triggered by climate variability on giant clam population management in the Pacific Ocean[END_REF]. Given such knowledge deficiencies, we aimed herein to characterize the physiological and transcriptomic responses of clams and their symbionts to hypothetically sublethal elevated temperatures (~30.7°C over a two-month period) that aimed to mimic past episodes of anomalously high temperatures in French Polynesia. In addition to hypothesizing that the giant clams would ultimately acclimate to this experimentally elevated temperature, we further hypothesized that a "dualcompartmental" bioinformatic approach, similar to the one that has been used with corals [START_REF] Mayfield | Compartment-specific transcriptomics in a reef-building coral exposed to elevated temperatures[END_REF], would provide insight into the key molecular pathways underlying the ability of each member of this association to acclimate to an environmentally relevant, sub-lethal temperature.

Materials and Methods

Experimental design, tissue sampling, and physiological measurements

The experimental procedures were first described by Brahmi et al. (2019). Briefly, 24 individual clams (N=4/treatment) were sampled over a 65-day period (days 29, 53, and 65) in control (29.2°C; ambient at the time of experimentation) and elevated (30.7°C) temperature conditions. The temperatures employed and the duration of the experiment reflect conditions in normal and abnormally hot seasons, respectively [(correlated with mass clam bleaching events [START_REF] Addessi | Giant clam bleaching in the lagoon of Takapoto atoll (French Polynesia)[END_REF]] reported in lagoons of French Polynesia's Tuamotu region (Brahmi et al., 2019). Samples (approx. 1 cm 2 ) from each of the two treatments at each of the three sampling times were systematically collected from the same region of the mantle and stored in RNALater® (Life Technologies, USA) at -80°C until analysis (N=24). Furthermore, a single hermaphroditic individual (approximately two years old) was sampled for a total of seven different tissues (mantle, adductor muscle, gonads, gills, byssus, visceral mass, and kidney) for transcriptome assembly. Only one individual was used in an effort to reduce assembly polymorphism biases. For this individual, which was excluded from the quantification analysis outlined below, sexual status was confirmed by gonad biopsy and histology following a previously detailed procedure [START_REF] Menoud | Identifying robust proxies of gonad maturation for the protandrous hermaphrodite Tridacna maxima (Röding, 1798, Bivalvia) from individual to population scale[END_REF].

Additionally, 10 giant clams were collected in situ in October 2018 around Reao Atoll (Tuamotu Archipelago, French Polynesia); tissues from each of these in situ individuals were collected from the same region of the mantle (approx. 1 cm 2 ) and stored in 95% ethanol at -20°C until later symbiont community analysis (described below).

As described in detail in Brahmi et al. (2019), a variety of physiological response variables were assessed in the 24 experimental replicates, in addition to the profiling of their transcriptomes: growth, Symbiodiniaceae density, and the maximum dark-adapted yield of photosystem II (Fv/Fm; as measured by an AquaPen pulse amplitude modulating fluorometer; APC-100m, Photon System Instruments, Czech Republic). Please see Brahmi et al. (2019) for details on these analyses. Physiological data were tested with two-way ANOVA (treatment x time) followed by Tukey's "honestly significant difference" (HSD) post-hoc tests (p<0.05), including the interaction between time and temperature, when data (raw or transformed) met the assumptions for ANOVA. For Symbiodiniaceae density and Fv/Fm, a non-parametric equivalent of the two-way ANOVA, the Scheirer-Ray-Hare test, was instead used (followed by Dunn's post-hoc tests).

DNA/RNA extractions and transcriptome sequencing

Total RNA was extracted from T. maxima mantles by lacerating tissues with a scalpel and rinsing with 1X PBS.

Cellular lysis was induced by addition of 1.5 ml TRIzol (Invitrogen, USA) according to the manufacturer's recommendations. The supernatant was transferred into a 2-ml tube and incubated for 10 min on ice. Phase separation was achieved by addition of 300 μl of chloroform coupled with centrifugation at 12,000 xg for 12 min at 4°C. The upper aqueous layer contained the RNA, and the lower organic layer was stored at -20°C for later DNA extraction (according to the manufacturer's recommendations). Total RNA from each individual was subjected to a DNAse treatment using Qiagen's RNA cleanup kit (Germany). RNA and DNA were quantified using a NanoDrop ND-2000 spectrophotometer (Thermo-Fisher, USA), and RNA quality was further evaluated by a Bioanalyzer 2100 (Agilent, USA). High-quality RNA was sent to McGill University's "Genome Quebec Innovation Center" (Montréal, QC, Canada) for Nextera XT (Illumina; USA) library preparation and sequencing on an Illumina HiSeq4000 100-bp paired-end platform. Samples for transcriptome assembly (N=7) were sequenced on a single lane, while samples for expression level quantification analysis (N=24) were uniformly and randomly distributed over two sequencing lanes after barcoding.

Transcriptomes assembly

Raw reads provided by RNA-Seq were filtered for quality and length using Trimmomatic v.0.36 [START_REF] Bolger | Trimmomatic: a flexible trimmer for Illumina sequence data[END_REF] with minimum length, trailing, and leading quality parameters set to 60 bp, 20, and 20, respectively. Illumina's adaptors and residual cloning vectors were removed via the UNIVEC database (https://www.ncbi.nlm.nih.gov/tools/vecscreen/univec/). Paired-end filtered reads were assembled de novo using Trinity v2.6.6 [START_REF] Haas | De novo transcript sequence reconstruction from RNA-seq using the Trinity platform for reference generation and analysis[END_REF] with a default k-mer size of 25 bp and a minimum transcript length of 200 bp. Raw transcripts (n=726,689; 420 Gbp) were filtered for presence of open reading frames (ORFs) (length≥300 bp), longest isoform matches, and mapping rate (≥0.5 transcripts per million; TPM).

Transcripts matching Refseq entries from archaea, plasmids, viruses, and bacteria (BLASTn; evalue<10 -10 ), as well those transcripts that aligned significantly (e-value<10 -4 ) only to bacterial sequences in the NCBI nt database (max target seqs=5) were discarded in an effort to reduce putative contamination. To segregate between symbiont and host sources, the meta-transcriptome was blasted (BLASTn; e-value<10 -4 ) against a pool of Symbiodiniaceae genomes and transcriptomes including former clades A, C, and F [sensu [START_REF] González-Pech | Signatures of adaptation and symbiosis in and transcriptomes of Symbiodinium[END_REF]]. By default, all hits with no match were considered as originating from the host. For quality control, the de novo transcriptome's completeness was assessed with BUSCO's v2 metazoa and v2 eukaryotes databases for clam and Symbiodiniaceae, respectively [START_REF] Simão | BUSCO: assessing genome assembly and annotation completeness with single-copy orthologs[END_REF]. Transcriptomes were annotated by BLAST search against the Uniprot-Swissprot database (BLASTx; e-value<10 -4 ). A schematic representation of the overall analysis pipeline has been provided in the Github repository (https://github.com/jleluyer/acclimabest).

Compartment-specific responses of the clam-dinoflagellate holobiont to long-term temperature exposure

Filtered reads were mapped against a combined host-symbiont transcriptome using GSNAP v2018.07.04 [START_REF] Wu | GMAP and GSNAP for genomic sequence alignment: enhancements to speed, accuracy, and functionality[END_REF] using the default parameters but allowing for a maximum mismatch value of 3 and a minimum coverage of 0.85. Only properly paired and uniquely mapped reads were conserved for downstream analysis ("concordant_uniq;" [START_REF] Wu | GMAP and GSNAP for genomic sequence alignment: enhancements to speed, accuracy, and functionality[END_REF]. Gene counts were conducted with HTSEQ v0.11.2 [START_REF] Anders | HTSeq-a Python framework to work with high throughput sequencing data[END_REF] using the default parameters. A filtering step including removal of genes with residual expression >1 count per million (CPM) in 4 individuals was applied, and data were transformed using the "rlog" function (betaPriorVar=2) implemented in the DESeq2 v1.23.10 R package [START_REF] Love | Moderated estimation of fold change and dispersion for RNA-seq data with DESeq2[END_REF] for host and symbionts separately.

Signed co-expression networks were built for the host and symbiont datasets independently using the R package WGCNA with a filtering step for minimum overall variance (>10%) following the recommendations of [START_REF] Langfelder | WGCNA: an R package for weighted correlation network analysis[END_REF]. The main goal of this analysis was to cluster genes in modules correlated with time, temperature, and relevant physiological responses (Figure 1). Briefly, we fixed "soft" threshold powers of 6 and 11 for the host and symbiont datasets, respectively, using the scale-free topology criterion to reach a model fit (|R|) of 0.90 and 0.80, respectively. The modules were defined using the "cutreeDynamic" function (minimum of 50 genes by module and default cutting-height=0.99) based on the topological overlap matrix, and an automatic merging step with the threshold fixed at 0.25 (default) allowed us to merge correlated modules. For each module, we defined the module membership (kME; Eigengene-based connectivity), and only statistically significant (p<0.05) modules were conserved for downstream functional analysis (Figure 1). Gene ontology (GO) enrichment analyses were conducted for each module using the GO_MWU R package [START_REF] Wright | Gene expression associated with white syndromes in a reef building coral, Acropora hyacinthus[END_REF] based on the background gene dataset found in WGCNA. GO terms were considered enriched at Benjamini-Hochberg adj. p<0.05 (minimum of three genes for any individual GO term).

Meta-analysis of cultured and in hospite Symbiodiniaceae transcriptomes

We integrated publicly available datasets featuring similar experimental designs (i.e. control and elevated temperature conditions over a long-term timescale) to further unravel conserved symbiont responses across genera, holobionts, and culture environments (i.e., cultured vs. in hospite). Manuscript searches were conducted with the Web of Science platform using the search formula: «symbio* AND RNAseq* AND temperature» together with informal searches via other research engines (e.g., Google Scholar). A total of three studies met our criteria: [START_REF] Levin | Sex, scavengers, and chaperones: transcriptome secrets of divergent Symbiodinium thermal tolerances[END_REF] and [START_REF] Gierz | Transcriptomic analysis of thermally stressed Symbiodinium reveals differential expression of stress and metabolism genes[END_REF] for cultured Symbiodiniaceae (n=48 transcriptomes) and [START_REF] Mayfield | Compartment-specific transcriptomics in a reef-building coral exposed to elevated temperatures[END_REF] for the response of Symbiodiniaceae in hospite with the scleractinian coral P. damicornis (n=12 transcriptomes). [START_REF] Gierz | Transcriptomic analysis of thermally stressed Symbiodinium reveals differential expression of stress and metabolism genes[END_REF] exposed cultured Symbiodiniaceae (Fugacium kawagutii; formerly clade F) to a 31°C heat stress (control temperature=24.5°C) over a 28-day period, while [START_REF] Levin | Sex, scavengers, and chaperones: transcriptome secrets of divergent Symbiodinium thermal tolerances[END_REF] exposed Symbiodiniaceae (Cladocopium goreaui; formerly type C1; including established thermo-tolerant and thermosensitive phenotypes) to a 32°C heat stress (control temperature=27°C) over a 13-day period. Finally, [START_REF] Mayfield | Compartment-specific transcriptomics in a reef-building coral exposed to elevated temperatures[END_REF] exposed corals housing Symbiodiniaceae (Cladocopium spp.; formerly a mixed assemblage of clade C individuals) to 30°C over a 9-month period (control temperature=27°C), and both the coral hosts and in hospite Symbiodiniaceae appeared to have acclimated to this temperature.

Raw data processing followed the same procedure as described above, though adapted for single-end reads for cultured Symbiodiniaceae datasets. To explore the convergence of Symbiodiniaceae responses despite large phylogenetic differences across the Symbiodiniaceae genera (Symbiodinium, Cladocopium, and Fugacium;[START_REF] Lajeunesse | Systematic Revision of Symbiodiniaceae Highlights the Antiquity and Diversity of Coral Endosymbionts[END_REF], we first searched for singlecopy orthologs across the three genera using OrthoFinder v2.2.7 [START_REF] Emms | OrthoFinder: solving fundamental biases in whole genome comparisons dramatically improves orthogroup inference accuracy[END_REF] based on publicly available genomes (http://reefgenomics.org/; [START_REF] Liu | Symbiodinium genomes reveal adaptive evolution of functions related to coraldinoflagellate symbiosis[END_REF]. We found a total of 4,215 ortho-groups that were used for downstream analyses. The count matrix was filtered for low residual expression genes (>1 CPM in 40 individuals; 4,187 remaining genes), and raw count data were transformed using the "vst" function implemented in the DESeq2 R package [START_REF] Love | Moderated estimation of fold change and dispersion for RNA-seq data with DESeq2[END_REF]. We used the "removeBatchEffect" function implemented in the Limma R package [START_REF] Ritchie | limma powers differential expression analyses for RNA-sequencing and microarray studies[END_REF] to remove experimental effects and fit the data prior the downstream analyses.

We then used a combination of redundant discriminant analysis (RDA) and partial dbRDAs approaches to assess the effect of temperature across Symbiodiniaceae clades and experiments. First, we computed a Euclidian distance matrix and performed a principal coordinates analysis (PCoA) on this Euclidian distance matrix using the "daisy" and "pcoa" functions, respectively, implemented in the "ape" R package [START_REF] Paradis | APE: Analyses of Phylogenetics and Evolution in R language[END_REF]. Only PCo axes explaining at least 2.5% of the total variance were kept for downstream analysis [START_REF] Legendre | Ecologically meaningful transformations for ordination of species data[END_REF]Legendre & legendre, 2012). To test for the effect of temperature and time, a distance-based redundancy analysis (db-RDA) was also produced with the retained PCo factors (n=8) as a response matrix and the variables temperature, experiment, and time as the explanatory factors. We first carried out stepwise model selection to identify relevant explanatory variables using the "ordistep" function implemented in the vegan R package [START_REF] Oksanen | Vegan: community ecology package[END_REF] and ultimately retained only temperature and time (p<0.05). Partial db-RDAs were therefore produced to test for the effects of these two parameters alone (no effect of experiment or genotype) after constraining the remaining variables. The effect of a given factor was considered significant when p<0.05. Finally, we used a weighted co-expression network analysis with WGCNA (similar thresholds as described above but with soft power fixed at 14) to reach a model fit (|R|) of 0.83, and subsequent module-wise GO enrichment analyses were undertaken using the GO_MWU R package [START_REF] Wright | Gene expression associated with white syndromes in a reef building coral, Acropora hyacinthus[END_REF].

Genomic basis of thermotolerance in Symbiodiniaceae dinoflagellates

We used an independent WGCNA co-expression network analysis to search for specific gene modules correlated with thermotolerance. For this purpose we focused on the dataset of [START_REF] Levin | Sex, scavengers, and chaperones: transcriptome secrets of divergent Symbiodinium thermal tolerances[END_REF], with Cladocopium goreaui as the reference genome [START_REF] Liu | Symbiodinium genomes reveal adaptive evolution of functions related to coraldinoflagellate symbiosis[END_REF]. Indeed, this is the only study to our knowledge featuring established thermotolerant phenotypes with transcriptomic data on long-term time series. The WGCNA analysis followed similar steps as described previously based, though based on rlog-transformed data (betaPrior=2). The soft threshold power was fixed at 20 to reach a model fit (|R|) of 0.85. The downstream, module-wise GO enrichment analyses followed the pipeline outlined above. Finally, we used the 'GO_deltaRanks_correlation' function implemented in the GO_MWU R package [START_REF] Wright | Gene expression associated with white syndromes in a reef building coral, Acropora hyacinthus[END_REF] to assess similarity between response to stress in symbiont in hospite with clams in and specific mechanisms of thermotolerance for cultured Symbiodiniaceae.

Results

Quantitative PCR-and meta-barcoding-based Symbiodiniaceae analysis

We evaluated the relative levels of various Symbiodiniaceae genera in our clam samples using a series of quantitative PCR (qPCR) assays. Amplifications were carried out on AriaMx real-time PCR System (Agilent, USA) using six primer sets optimized for the amplification of nuclear ribosomal 28S in Symbiodiniaceae of clades/genera A-F [START_REF] Yamashita | Do corals select zooxanthellae by alternative discharge?[END_REF] following the protocol of [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]. The PCRs (25 µL) comprised 12.5 µL of 2X SYBR® Green master mix (Agilent, USA), 10 µL of DNA (previously diluted to 1 ng µL -1 ), and 1.25 µL of each primer (forward and reverse; each at a stock concentration of 4 µM). PCR thermocycling included:

1 cycle of pre-incubation for 10 min at 95°C; 40 cycles of amplification (30 s at 95°C, 1 min at 64°C, and 1 min at 72°C), and a melting curve analysis that extended from 60°C to 95°C (30-s incubations). All measurements were made in duplicate, and all analyses were based on the threshold cycle (Ct) values of the PCR products.

Ct values were averaged across duplicate samples when the variation was not exceeding 1; otherwise, samples were re-run until delta Ct<1. Similarity in relative clade abundance was assessed using PCA analysis of a Bray-Curtis similarity matrix with Hellinger-transformed data. Db-RDAs were conducted to identify whether either temperature or time had a significant impact on Symbiodiniaceae assemblage, and an alpha level of 0.05 was set a priori. To complement data from the experimental individuals, qPCRs were carried out with DNA isolated from mantle fragments from the 10 wild individuals described above collected from Reao Atoll [geographically proximal to the origin of the experimental individuals; see Brahmi et al., (2019) for details.] in October 2018. Sample preparation and analyses were performed as described above and in [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].

As a more detailed means of assessing Symbiodiniaceae diversity in the 24 clam samples, a meta-barcoding analysis was undertaken following the protocol of [START_REF] Cunning | Using high-throughput sequencing of ITS2 to describe Symbiodinium metacommunities in St. John, U.S. Virgin Islands[END_REF]. Briefly, the ITS2 gene was PCR amplified using previously described primers [START_REF] Cunning | Using high-throughput sequencing of ITS2 to describe Symbiodinium metacommunities in St. John, U.S. Virgin Islands[END_REF]) and sequenced at the facility listed above, albeit on a Illumina Miseq 250bp paired-end platform. The Dada2 algorithm [START_REF] Callahan | DADA2: High resolution sample inference from Illumina amplicon data[END_REF] implemented in the QIIME2 software package [START_REF] Bokulich | Optimizing taxonomic classification of marker-gene amplicon sequences with QIIME 2's q2-feature-classifier plugin[END_REF] was used to infer exact sample sequences from amplicon data. The reference database was directly imported from the NCBI nt repository and trained on the basis of the ITS2 primers following [START_REF] Cunning | Using high-throughput sequencing of ITS2 to describe Symbiodinium metacommunities in St. John, U.S. Virgin Islands[END_REF]

Physiology

We observed no mortality across the 65-day experiment, but some of the individuals exposed to elevated temperature showed signs of partial bleaching in the 30.7°C treatment by day 65. Symbiodiniaceae density and photosynthetic yield (Fv/Fm) were both lower in clams exposed to elevated temperatures (Scheirer-Ray-Hare;

H=24.44, p<0.001 and H=22.88, p<0.001, respectively; Figure S1). There was no interaction between time and temperature for Symbiodiniaceae Fv/Fm (Scheirer-Ray-Hare; H=1.26; p=0.53, Figure S1). Time had only a slight effect on Symbiodiniaceae density (Scheirer-Ray-Hare; H=6.07; p=0.048, Figure S1), though no post-hoc differences were detected between individual sampling times (Dunn's test; p>0.05).

Symbiodiniaceae communities in hospite with clams

The Symbiodiniaceae communities of all clam hosts (from both control and high temperature conditions) were primarily composed of Symbiodinium spp. (formerly clade A; Figure 1A). F=1.3; q-value=0.33) nor evenness values (Kruskall-Wallis; H=0.04; q-value=0.83) differed significantly between temperatures.

Transcriptome assemblies

A total of 363.70 million 100-bp paired-end reads were used to assemble a raw metatranscriptome (host + symbionts) of 726,689 transcripts (420.02 Gbp). After stringent filtering and segregation of host and Symbiodiniaceae sequences, the assemblies resulted in a transcriptome for T. maxima of 24,234 contigs (N50=1,011 bp; GC content=40.1%) and a meta-transcriptome for Symbiodiniaceae of 51,648 contigs (N50=1,027 bp; GC content=57.9%). High G-C content is generally a hallmark of Symbiodiniaceae transcriptomes [START_REF] González-Pech | Signatures of adaptation and symbiosis in and transcriptomes of Symbiodinium[END_REF]. Transcriptome statistics and annotations are provided in Figure 2 and Table S1, respectively.

Host clam acclimation response to prolonged high-temperature exposure

A gene co-expression network was built using the normalized RNA-Seq data from which lowexpression genes had been eliminated, and three modules correlated significantly (p<0.05) with temperature and/or physiological data (including oxygen production, Symbiodiniaceae density and Fv/FM, and host dry weight; Figure S2). No module was correlated with sampling time, O2 consumption, or shell extension. A single host module (pinkhost)

positively correlated with temperature (R=0.82) and negatively with photosynthetic rate and symbiont density (R=-0.52 and R=-0.48, respectively; Figure S2). The redhost module also correlated positively with Fv/Fm (R=0.59) but not significantly with temperature (R=-0.38; p=0.08). Among the most enriched GO terms in the pinkhost module were pituitary gland development (GO:0021983), L-ascorbic acid metabolic processes (GO:0019852), regulation of extrinsic apoptotic signaling pathways (GO:2001236), cholesterol efflux (GO:0033344), cilium movement (GO:0003341), and ommochrome biosynthetic processes (GO:0006727). Ommochromes are biological pigments and metabolites of tryptophan [START_REF] Figon | Ommochromes in invertebrates: biochemistry and cell biology: Ommochromes in invertebrates[END_REF]. The redhost module was enriched for cation transport (GO:0006812), neurotransmitter uptake (GO:0001504), fructose 6-phosphate metabolic processes (GO:0006002), and reactive oxygen species metabolic processes (GO:0072593). Host module membership eigenvalues were also integrated with the symbiont network analysis (Figure 3), and a complete list of GO-enriched functions has been provided in Table S2.

Acclimation to prolonged high-temperature exposure in Symbiodiniaceae in hospite with clams

Co-expression network analysis of Symbiodiniaceae showed more modules correlated with temperature than for the clam host, either negatively [midnightbluesymbiont (R=-0.94), bluesymbiont (R=-0.45)] or positively

[cyansymbiont (R=0.61), blacksymbiont (R=0.91), yellowsymbiont (R=0.52), and pinksymbiont (R=0.85); Figure 3]. Among the enriched GO terms in the blacksymbiont module were RNA processing (GO:0006396), methylation (GO:0043414), chloroplast-nucleus signaling pathways (GO:0031930), and glycerolipid metabolic processes (GO:0046486). For the cyansymbiont module, enriched GO terms included response to vitamins (GO:0033273), response to UV-C (GO:0071494), regulation of transferase activity (GO:0051338), intrinsic apoptotic signaling pathways (GO:0097193), and induced systemic resistance (GO:0009682). The yellowsymbiont module featured RNA modification (GO:0009451) and aspartate family amino acid metabolic processes (GO:0009066). Finally, the bluesymbiont module showed enrichment for movement of cellular or subcellular components (GO:0006928), reproduction (GO:0000003), regulation of cell shape (GO:0008360), oxidation-reduction processes (GO:0055114), and electron transport chain (GO:0022900) while the midnightbluesymbiont module featured enrichment for regulation of BMP signaling pathways (GO:0030510), hormone biosynthetic processes (GO:0042446), peptidyllysine dimethylation (GO:0018027), short-term memory (GO:0007614), and response to red or far, red light (GO:0009639). The complete GO enrichment results can be found in Table S2.

Multivariate analysis of public Symbiodiniaceae datasets

We used db-RDA to document gene expression variation in public Symbiodiniaceae datasets [in culture and in hospite with corals and clams (this study)], with temperature and time as the explanatory variables; there was a focus on single-copy orthologs from the genera Cladocopium, Fugacium, and Symbiodinium. The overall model was significant (p<0.001), and the adjusted R 2 was 0.12 (Figure 4). Partial db-RDAs showed that temperature also had a significant effect on total gene expression variation across genotypes and experiments (1000 permutations ;

F=9.07, p=0.001). A WGCNA analysis was conducted to identify genes cluster correlated with temperature across all the orthologous genes (Figure S3).

Search for thermotolerance-specific genes clusters

We also conducted independent WGCNA analyses to assess acclimatory responses in cultured Symbiodiniaceae based on the Cladocopium goreaui (formerly type C1) genome [START_REF] Liu | Symbiodinium genomes reveal adaptive evolution of functions related to coraldinoflagellate symbiosis[END_REF]) and compared them with thermotolerant phenotypes [START_REF] Levin | Sex, scavengers, and chaperones: transcriptome secrets of divergent Symbiodinium thermal tolerances[END_REF]. No individual module correlated with time. Instead, we found the majority of the genes to be correlated with temperature, and three modules were uncovered: darkgreyC1 (R=0.82), saddlebrownC1 (R=-0.89; N=1,354), and orangec1 (R=-0.87; N=378; Figure S4). We also found three modules (darkolivegreenC1, lightgreenC1, and whiteC1) that were significantly correlated with thermotolerance (R=-0.74, -0.99, and 0.98, respectively; Figure S4) but not temperature. These modules effectively differentiated thermosensitive Symbiodiniaceae from thermotolerant C1 phenotypes described in [START_REF] Levin | Sex, scavengers, and chaperones: transcriptome secrets of divergent Symbiodinium thermal tolerances[END_REF]. Among the most enriched GO terms for lightgreenc1 were cellular response to amino acid stimulus (GO:0071230), DNA methylation (GO:0006306), and genetic imprinting (GO:0071514; Figure S4 and Table S2). Furthermore, we found that impact on methylation-associated biological processes [methylation (GO:0032259) and macromolecule methylation (GO:0043414)] was conserved in the lightgreenC1 module and the response to temperature of symbionts in hospite with clams (blacksymb module; Figure S5).

Discussion

Temperature increases are threatening marine invertebrate populations worldwide, especially for species already living at, or close to, their upper thermal tolerance limits [START_REF] Hoffmann | Climate change and evolutionary adaptation[END_REF]. Recent heat wave events have resulted in ~90% declines in T. maxima populations in some atolls of French Polynesia [START_REF] Andréfouët | Climate variability and massive mortalities challenge giant clam conservation and management efforts in French Polynesia atolls[END_REF][START_REF] Andréfouët | Adaptive management for the sustainable exploitation of lagoon resources in remote islands: lessons from a massive El Niño-induced giant clam bleaching event in the Tuamotu atolls (French Polynesia)[END_REF]. While several studies have investigated the invertebrate (mollusc and cnidarian) response to heat stress over short-term timescales, relatively few have investigated the prolonged response to elevated temperatures (e.g., [START_REF] Mayfield | Compartment-specific transcriptomics in a reef-building coral exposed to elevated temperatures[END_REF]. Although our clam samples ultimately acclimated to an experimentally elevated temperatures of nearly 31°C, Symbiodiniaceae density was reduced in thermally challenged clams, and both host clams and their Symbiodiniaceae populations underwent gene expression changes over the course of this two-month experiment. Upon discussing such temperature-driven changes in gene expression, we highlight some intrinsic responses of the symbionts (i.e., independent of the host species) and identify key mechanisms potentially underlying their thermal-tolerance.

Genus-specific fidelity in clam hosts might preclude symbiont community shifts/shuffling as a thermal acclimation strategy A 1.5°C temperature elevation over a 65-day period was sufficient to induce a significant reduction in symbiont density in clams; no bleaching (even partial) was observed in control temperature clams. Our results support previous studies of corals and giant clams in which high-temperature exposure led to sub-lethal bleaching [START_REF] Ainsworth | Early cellular changes are indicators of pre-bleaching thermal stress in the coral host[END_REF]Brahmi et al., 2019;[START_REF] Hoegh-Guldberg | The effect of sudden changes in temperature, light and salinity on the population density and export of zooxanthellae from the reef corals Stylophora pistillata Esper and Seriatopora hystrix Dana[END_REF][START_REF] Jones | Temperature-induced bleaching of corals begins with impairment of the CO2 fixation mechanism in zooxanthellae[END_REF][START_REF] Leggat | The impact of bleaching on the metabolic contribution of dinoflagellate symbionts to their giant clam host[END_REF][START_REF] Warner | Damage to photosystem II in symbiotic dinoflagellates: A determinant of coral bleaching[END_REF][START_REF] Zhou | Oxidative stress, apoptosis activation and symbiosis disruption in giant clam Tridacna crocea under high temperature[END_REF]; whether the cellular mechanisms of bleaching are conserved between corals and giant clams remains to be determined (Mies et al., 2017;[START_REF] Zhou | Oxidative stress, apoptosis activation and symbiosis disruption in giant clam Tridacna crocea under high temperature[END_REF].

For some coral species, resilience to heat stress is associated with a more flexible symbiotic association (i.e., the capacity to shift from one dominant Symbiodiniaceae genus to another) [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][START_REF] Lajeunesse | High diversity and host specificity observed among symbiotic dinoflagellates in reef coral communities from Hawaii[END_REF][START_REF] Putnam | Endosymbiotic flexibility associates with environmental sensitivity in scleractinian corals[END_REF][START_REF] Rowan | Thermal adaptation in reef coral symbionts[END_REF][START_REF] Silverstein | Specificity is rarely absolute in coral-algal symbiosis: implications for coral response to climate change[END_REF]. Indeed, some bleaching events have largely been attributed to the thermal sensitivity of specific endosymbiotic Symbiodiniaceae residing in coral host tissues [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][START_REF] Oliver | Many corals host thermally resistant symbionts in hightemperature habitat[END_REF]. Corals hosting Cladocopium spp. (formerly clade C) are typically more prone to bleaching, whereas those housing certain lineages of Durusdinium (formerly clade D) have demonstrated enhanced thermotolerance [START_REF] Baker | Flexibility and Specificity in Coral-Algal Symbiosis: Diversity, Ecology, and Biogeography of 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]. Interestingly, Cladocopium spp. and/or Durusdinium spp. are more commonly found in giant clams inhabiting warmer environments while Symbiodinium spp. (formerly clade A) are more common in clams located in cooler waters [START_REF] Deboer | Patterns of Symbiodinium distribution in three giant clam species across the biodiverse Bird's Head region of Indonesia[END_REF]. Herein, the Symbiodiniaceae communities were predominantly composed of Symbiodinium spp., even after two months of high-temperature exposure; this finding aligns with other studies in corals that found Symbiodiniaceae assemblages to be temporally stable, even as environmental conditions changed [START_REF] Goulet | Most corals may not change their symbionts[END_REF][START_REF] Sampayo | Bleaching susceptibility and mortality of corals are determined by fine-scale differences in symbiont type[END_REF][START_REF] Thornhill | Multi-year, seasonal genotypic surveys of coral-algal symbioses reveal prevalent stability or post-bleaching reversion[END_REF][START_REF] Thornhill | Reef Endemism, Host Specificity and Temporal Stability in Populations of Symbiotic Dinoflagellates from Two Ecologically Dominant Caribbean Corals[END_REF]. This was not an artifact due to the experimental conditions enacted since individuals sampled from their original locations in situ also predominantly host Symbiodinium spp. (i.e., clade A).

Such a high proportion of Symbiodinium spp. in giant clams was expected, and it has also been reported in the sea anemone Anemonia viridis; however, it is in sharp contrast with other invertebrate hosts such as corals, which host a broader Symbiodiniaceae diversity [START_REF] Manning | Diversity in populations of free-living Symbiodinium from a Caribbean and Pacific reef[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]Stat, Carter, & Hoegh-Guldberg, 2006). This near-exclusive hosting of Symbiodinium spp. in clams, and the temporal stability of their association, suggests that some selection process favors this dinoflagellate lineage (or else impairs recruitment of others); lectin/glycan interactions were once thought to play a role, possibly in the primary recognition-related processes (WoodCharlson, Hollingsworth, Krupp, & Weis, 2006), though this hypothesis has recently been called into question [START_REF] Parkinson | Subtle Differences in Symbiont Cell Surface Glycan Profiles Do Not Explain SpeciesSpecific Colonization Rates in a Model Cnidarian-Algal Symbiosis[END_REF]

Effect of prolonged exposure to elevated temperature on the clam transcriptome

Both host clam and Symbiodiniaceae gene expression were affected by elevated temperature exposure, with no significant effects of time from 29 days onwards; the temperature-related differences were from thenceforth sustained over time. We found one gene module positively impacted by temperature and negatively correlated with symbiont Fv/Fm and density. This module showed enrichment for ommochrome biosynthesis process and specifically included the tryptophan 2,3-dioxygenase coding gene (TDO), a pivotal regulator of systemic tryptophan levels also involved in the response to oxidative stress [START_REF] Forrest | Tryptophan Loading Induces Oxidative Stress[END_REF][START_REF] Thackray | Exploring the mechanism of tryptophan 2,3-dioxygenase[END_REF].

Tryptophan is the precursor of 5-hydroxytryptamine (5-HT), a bivalve serotonin transmitter that plays critical roles in numerous physiological functions [e.g., reproduction [START_REF] Alavi | Structure-function of serotonin in bivalve molluscs[END_REF]]. In larvae from the coral Orbicella faveolata, TDO (referred to as AGAP) was upregulated in response to ultraviolet radiation, and larval fitness (locomotion and settlement) went on to suffer [START_REF] Aranda | Differential sensitivity of coral larvae to natural levels of ultraviolet radiation during the onset of competence[END_REF]. A more thorough understanding, then, of ommochrome biosynthesis and, more generally, tryptophan regulation, is likely to be key to elucidating the molecular regulation of invertebrate-dinoflagellate symbioses, nearly all of which involve at least some degree of nitrogen transfer within holobionts [START_REF] Chan | Light exposure enhances urea absorption in the fluted giant clam, Tridacna squamosa, and up-regulates the protein abundance of a light-dependent urea active transporter, DUR3-like, in its ctenidium[END_REF].

A single module was 1) positively correlated with the maximum dark-adapted yield of photosystem II (Fv/Fm) and 2) enriched for genes encoding proteins involved in glyceraldehyde-3phosphate metabolic processes. Glycerol excretion from dinoflagellate symbionts is largely influenced by the presence of host tissues [START_REF] Muscatine | Glycerol excretion by symbiotic algae from corals and Tridacna and its control by the host[END_REF].

The glyceraldehyde-3-phosphate pathway, which culminates in glycerol production, was also significantly affected by sub-lethal elevated temperature (30°C) exposure in the reef coral P. damicornis [START_REF] Mayfield | Compartment-specific transcriptomics in a reef-building coral exposed to elevated temperatures[END_REF]. Pollutant exposure also altered the expression of genes involved in carbohydrate metabolism, albeit only in the coral host compartment (and not in Symbiodiniaceae) in another study [START_REF] Gust | Coralzooxanthellae meta-transcriptomics reveals integrated response to pollutant stress[END_REF]. Admittedly, we did not assess the proportion of energy derived from autotrophy herein, which ranges widely (from 25 to up to 100%) and is dependent on the species and/or life history stage in the Tridacna genus [START_REF] Fisher | Photosynthesis and respiration in Tridacna gigas as function of irradiance and size[END_REF][START_REF] Klumpp | Contributions of phototrophic and heterotrophic nutrition to the metabolic and growth requirements of four species of giant clam (Tridacnidae)[END_REF][START_REF] Klumpp | Contributions of phototrophic and heterotrophic nutrition to the metabolic and growth requirements of four species of giant clam (Tridacnidae)[END_REF]; shifts from autotrophy to heterotrophy, and vice versa, are likely to affect host gene expression patterns. All that can be stated at present is that regulation of tryptophan levels and impairment of carbohydrate metabolism might be key elements in the long-term response to elevated temperature in clams; indeed, these two processes could be inter-linked. However, how these changes would affect finescale interactions between the host and symbionts remains to be explored and should be the focus of future studies of clam-Symbiodiniaceae symbioses.

The response of Symbiodiniaceae dinoflagellates in hospite with clams to prolonged elevated temperature exposure Overall, gene clusters of Symbiodiniaceae showed positive correlation between expression levels and prolonged elevated temperature exposure, and some of the modules were also correlated with the lower Symbiodiniaceae Fv/Fm and cell densities documented at elevated temperatures. Other physiological studies have also shown that high temperatures lead to diminished photosynthetic yield in several clades of Symbiodiniaceae [START_REF] Grégoire | Photophysiological and thermal tolerance of various genotypes of the coral endosymbiont Symbiodinium sp. (Dinophyceae)[END_REF]. In terms of the RNA-Seq data, genes encoding proteins involved in nitrogen metabolism were significantly affected by high-temperature exposure, and this module correlated with host tryptophan dehydrogenase activity. Interestingly, this GO includes the salt-and drought-induced ring finger1

(SDIR 1)-coding gene known in plants to control abscisic acid (ABA) signal transduction [START_REF] Zhang | SDIR1 Is a RING Finger E3 Ligase That Positively Regulates Stress-Responsive Abscisic Acid Signaling in Arabidopsis[END_REF], a process that has never before been reported in Symbiodiniaceae. The phytohormone ABA and ROS regulating/modulating proteins are key molecular constituents involved in the capacity to acclimate to abiotic stressors, including oxidative stress tolerance in unicellular algae [START_REF] Lu | Phytohormones in microalgae: a new opportunity for microalgal biotechnology[END_REF]. Furthermore, up-regulation of ABA signaling genes is associated with a later increase in ABA biosynthesis in several plant species [START_REF] Vishwakarma | Abscisic acid signaling and abiotic stress tolerance in plants: a review on current knowledge and future prospects[END_REF]. The role of ABA signaling in the thermo-adaptation of Symbiodiniaceae dinoflagellates may consequently be a fruitful avenue for future research.

Herein we also found that expression of genes encoding certain components of the photosynthetic machinery, especially photosystem II (PSII), was dampened at elevated temperature. PSII integrity is vital for proper Symbiodiniaceae function, and PSII damage has been directly linked to bleaching in corals [START_REF] Warner | Damage to photosystem II in symbiotic dinoflagellates: A determinant of coral bleaching[END_REF]. It is noteworthy that the same gene module also included chloroplast thylakoid membrane rearrangement-related genes, which are used by Symbiodiniaceae and other photosynthetic organisms to cope with heat and high UV radiation [START_REF] Sharkey | Effects of moderate heat stress on photosynthesis: importance of thylakoid reactions, rubisco deactivation, reactive oxygen species, and thermotolerance provided by isoprene[END_REF][START_REF] Slavov | Super-quenching" state protects Symbiodinium from thermal stress -Implications for coral bleaching[END_REF]. Although the clam-dinoflagellate holobionts generally appeared to have acclimated to elevated temperatures over our two-month experiment (no large-scale bleaching), the Symbiodiniaceae communities, then, showed signs of intracellular stress given these gene expression changes, as well as the decreases in cell density and Fv/Fm. Whether or not these holobionts could have sustained an even longer exposure to ~31°C remains to be determined, though it is worth noting that, unlike in situ, clams were not fed in the aquaria. It is thus likely that clams allowed to feed both autotrophically and heterotrophically might, then, have an even superior capacity for high-temperature acclimation.

Conserved response to high temperatures across Symbiodiniaceae genera and molecular mechanisms linked to thermo-acclimation capacity

We documented a conserved response to long-term exposure to elevated temperature across Symbiodiniaceae genera based only on orthologous genes, which is noteworthy given the large evolutionary distance between genera [START_REF] Correa | Understanding diversity in coral-algal symbiosis: a clusterbased approach to interpreting fine-scale genetic variation in the genus Symbiodinium[END_REF][START_REF] Lajeunesse | Investigating the biodiversity, ecology, and phylogeny of endosymbiotic dinoflagellates in the genus Symbiodinium using the ITS region: in search of a "species" level marker[END_REF]. This common response, which transcended the host effect, included genes involved in regulation of the DNA damage response, wound healing and low-temperature responses, chromatin remodeling, mRNA splicing, regulation of lipid biosynthetic processes, and motile cilium assembly. Our results, however, most likely underestimate the molecular complexity of thermo-acclimation given our use of exclusively "single-to-single" orthologous genes. It is also possible that there are holobiont-specific responses that were not explored or detected herein with our bioinformatics approach. For instance, recent studies have shown that the Symbiodiniaceae diverged, in part, in relation to their capacity for synthesizing UVabsorbing mycosporine-like amino acids [START_REF] Shoguchi | Draft assembly of the Symbiodinium minutum nuclear genome reveals dinoflagellate gene structure[END_REF]. Furthermore, while UV-B radiation in cultured Symbiodiniaceae drastically reduces photosynthetic output, such is not always observed for cells in hospite with clams since the clam hosts produce UV-absorbing proteins [START_REF] Ishikura | UV-absorbing substances in zooxanthellate and azooxanthellate clams[END_REF].

We further explored basal differences within the Cladocopium genus that would differentiate the contrastingly thermotolerant phenotypes. We found that differences between thermotolerant phenotypes were driven by molecular pathways uncovered previously [START_REF] Levin | Sex, scavengers, and chaperones: transcriptome secrets of divergent Symbiodinium thermal tolerances[END_REF], including meiotic nuclear division and glutathione disulfide oxidoreductase activity; expression of genes involved in photosynthesis, cellular heat acclimation, and methylation programming also differed across gradients of thermotolerance. Regarding the latter, epigenetic landscape rearrangement has been shown to play a role in transgenerational inheritance of thermo-tolerance of various plant models [START_REF] Bruce | Stressful "memories" of plants: and possible mechanisms[END_REF]. Here, thermotolerance-associated modules generally did not correlate with temperature, suggesting that phenotypes have intrinsic gene expression signatures that respond differentially to changes in temperature. It is known that in plants DNA methylation and histone modification are associated with the response to heat stress, and, more specifically, act to prevent heatassociated macromolecular damage [START_REF] Liu | Genetic and epigenetic control of plant heat responses[END_REF]. Such methylation changes might be inherited and account for, at least in part, the remarkable ability of plants to adapt and/or acclimate quickly to stressful environments [START_REF] Ganguly | The Arabidopsis DNA methylome is stable under transgenerational drought stress[END_REF][START_REF] Lämke | Epigenetic and chromatin-based mechanisms in environmental stress adaptation and stress memory in plants[END_REF].

Conclusions

The co-expression network analysis proved to be a powerful tool for dissecting compartmentspecific transcriptomic responses in symbiotic systems. This is especially true when looking for acclimatory signatures that, in contrast to short-term stress responses, are characterized by rather subtle changes over longer periods. Indeed, our data from a long-term high temperature study revealed that different cellular processes are impacted in the host clam and in hospite Symbiodiniaceae compartments; genes encoding key photosynthesis proteins were particularly temperature sensitive in not only Symbiodiniaceae in hospite, but also in culture. Future studies focusing on the range of optimal thermal conditions of the T. maxima species may improve our understanding on the thermal tolerance of the clams and their symbionts. Although the giant clams used in this study ultimately survived a two-month exposure to nearly 31°C, it is possible that slightly higher temperatures, or extended exposure times, might cause them to bleach to such a great extent that they would not survive. Regardless, our data show that novel mechanisms involving epigenetic landscape rearrangement are associated with elevated Symbiodiniaceae thermotolerance. How the impact of stressful environmental conditions might impact the subsequent generation's tolerance and/or physiological capacities (i.e., epigenetic effects) must consequently be addressed in the near future.

  Four clams, however, were characterized by secondary populations of Cladocopium spp. (formerly clade C; with relative proportions reaching 1.8 to 32.8%), as well as residual quantities (<0.001%) of Breviolum (formerly clade B) and Fugacium (formerly clade F). There were no detectable effects of prolonged high-temperature exposure of the Symbiodiniaceae assemblages within the giant clam samples (Figure1B). Similarly, in situ clam samples from Reao Atoll were also dominated by Symbiodinium spp. (mean 93.0%±10.7 SD), with smaller populations of Breviolum spp. and Cladocopium spp. Given the similarities in Symbiodiniaceae assemblages between the experimental and in situ specimens, we conclude that transport out of the ocean and into the aquarium husbandry facility did not result in community changes that could bias the results described below.Metabarcoding of the internal transcribed spacer 2 (ITS2) sequence resulted in an average of 186.7k 25.7 PE sequences per sample. After sequence pre-processing, the Dada2 algorithm reported a total of 12 amplicon sequence variants matching to Symbiodinium spp. (N=9) and Cladocopium spp. (N=3) that paralleled results from qPCRs. Symbiodinium sequence variants mainly matched to S. tridacnidorum (formerly sub-clade A3; best-hit BLASTn e-value<10 -6 ). Neither cladal/genera representation based on UniFrac distance (PERMANOVA; pseudo-

  . Admittedly, broader in situ clam sampling, (e.g., encompassing different times of the year) will be necessary to verify the fidelity between Symbiodinium spp. and giant clams, and whether mixed-genera assemblages are common in situ (DeBoer et al., 2012; Parkinson, Banaszak, Altman, LaJeunesse, & Baums, 2015). The presumably low flexibility would appear to preclude community shifts as a strategy for these clams to cope with increased temperatures, at least in our experimental context. Rather than adaptation (i.e., a community shift resulting in a new "holobiont genomic landscape"), acclimation (i.e., physiological changes that initially manifested at the molecular level) appears to have played a larger role in this study.

Acknowledgments

General: We are grateful to Michel Pahuatini for providing the "small giant" clams and seawater samples. We thank Claude Soyez and Leila Chapron for their help in animal rearing. We thank Dr. Laetitia Hédouin for valuable comments on prior versions of the manuscript, Corinne Belliard for help with DNA preparation, and Dr. Yu-Bin Wang for making the P. damicornis-Symbiodiniaceae transcriptomic resources openly accessible, as well as designing the afore-cited P. damicornis-Symbiodiniaceae transcriptome server. Finally, we thank O.

Bichet for her help with the figures.

Funding: This experiment was made possible by a grant from Labex CORAIL (France), as well as an IFREMER grant (France; Master's internship to HAM).

Author contributions: CB and JVD conceived the experimental design, under which tissue samples featured herein were collected. CB and JLL conceived the transcriptomic study. HAM and JLL carried out the laboratory benchwork.

HAM and JLL analyzed the data. HAM, CB, ABM, and JLL wrote the manuscript. All co-authors contributed substantially to revised drafts of the manuscript. We also thank two anonymous reviewers for their help in largely improving the manuscript.

Competing interests:

We declare that we have no competing interests.

Data and materials availability: Raw sequencing RNA-Seq data for small giant clams featured herein have been made publicly available on the NCBI database (PRJNA579426), and all scripts discussed in the article can be found on Github (https://github.com/jleluyer/acclimabest). Raw meta-barcoding data are available here: (pending creation). Data


Jana Pisk

Dominique Agustin
email: dominique.agustin@iut-tlse3.fr

Višnja Vrdoljak

Rinaldo Poli

Epoxidation Processes by Pyridoxal Dioxomolybdenum(VI) (Pre)Catalysts Without Organic Solvent

Keywords: Epoxidation, Molybdenum, Pyridoxal ligands, Homogeneous catalysis, Solvent-free

Molybdenum(VI) pyridoxal thiosemicarbazonato complexes have proved to be efficient catalysts in olefin epoxidation of cyclooctene by aqueous TBHP in absence of organic solvent. High activity and selectivity have been obtained with a catalyst loading of just 0.05% molybdenum in the case of one mononuclear (pre)catalyst (TOF 3360 h -1 ) under solvent-free conditions. The influence of methanol on activity and selectivity has been studied.

Epoxides, as precious precursors in organic synthesis, can be easily obtained from alkenes by the use of strong organic oxidants (m-CPBA, NaClO) [1] or smoother oxidants (THBP, H2O2) [2] with the assistance of metal-based catalysts (Fe, Mn, Re, Mo, V, W). [2,3] Among these metals, molybdenum attracts most of the attention. [3][4][5][6] Different stable Mo VI complexes have been used as pre-catalysts. [2a,2b,3-6] Most of the investigated processes are performed in chlorinated solvents with catalyst loadings as high as 1% Mo. Solvent recovery asks extra efforts and costs that could be eliminated by the application of greener processes. Dioxomolybdenum(VI) complexes with tridentate Schiff base (ONO) ligands are interesting candidates because of their facile preparation, structural flexibility, and stability; several of such complexes have already been used as epoxidation (pre)catalysts. [4,5] On the other hand, tridentate ligands with an ONS donor set are much less common. There is apparently only one report of a catalyzed epoxidation process carried out with a Mo VI complex containing an ONS ligand. [6] In the aim of developing cleaner processes, [START_REF]Green Separation Processes: Fundamentals and Applications[END_REF] we have investigated a few recently published [START_REF] Vrdoljak | [END_REF] Mo VI (pre)catalysts containing pyridoxal-based ONS ligands for olefin epoxidation by aqueous TBHP, without addition of extra organic solvent. Emphasis is placed on the low Mo content (0.05% Mo vs. olefin) and the solvent-free operating conditions. Scheme 1. Mo(VI) pyridoxal thiosemicarbazonato complexes used as (pre)catalysts.

Pyridoxal thiosemicarbazonato ligands have been distinguished as a new class of ligands presenting antiviral and antitumoral activity when complexed to transition metals. [9] In the recently reported molybdenum complexes, [START_REF] Vrdoljak | [END_REF] these ligands coordinate the central metal atom in the doubly deprotonated form through the oxygen, sulfur and nitrogen atoms. Mononuclear [MoO2(L 1 )(MeOH)] and polynuclear [MoO2(L 1-3 )]n species (see Scheme 1) are available as previously described. [START_REF] Vrdoljak | [END_REF] The [MoO2(L 1 )]n polymer can be converted to the corresponding mononuclear [MoO2(L 1 )(MeOH)] when dissolved in MeOH, whereas the opposite transformation occurs upon treating the mononuclear methanol adduct with suitable solvent such as acetonitrile (Scheme 2). Stable MeOH adducts derived from the polymers containing the L 2 and L 3 ligands, on the other hand, could not be isolated. Although many previous contributions report on the catalytic activity of molybdenum species in alkene epoxidation, [3,4,10] only few deal with Mothiosemicarbazonato complexes. [6,11] Furthermore, the typical Mo:substrate ratio is 1:100 [12] and nearly all epoxidation reactions were performed in organic solvents. [2a-b,4,6,10,12-14] We have recently observed a solvent-free process using similar complexes (with a variety of ONO ligands), with high activity and high selectivity for the epoxidation of cyclooctene (for instance, up to 98% when using [MoO2(SAP)]2, SAP = N-salicylidene-2 aminophenolato), only when using a Mo:substrate ratio of 1:100. [5] With a lower Mo:substrate ratio (1:2000), the same [MoO2(SAP)]2 complex gave a substantially weaker selectivity (68%, see SI). Pyridoxal-based ONS Mo VI complexes have now been tested under the same conditions and with low 0.05% Mo catalyst loading and we herein communicate the corresponding results.

The investigated [MoO2(L 1 )(MeOH)] and [MoO2(L 1-3 )]n complexes are sparingly soluble in cyclooctene and insoluble in water at room temperature, but dissolve completely in the organic phase after addition of aqueous TBHP at 80 °C. The aqueous phase was colourless and the organic one yellowish, indicating that the catalyst is mainly confined in the organic phase. The epoxidation results are shown in Fig. 1.

The selectivity is very high (84-99%) for all tested molybdenum compounds, following the order

[MoO2(L 3 )]n ~ [MoO2(L 1 )(MeOH)] > [MoO2(L 2 )]n > [MoO2(L 1 )]n.
The epoxide yield is moderate to very good after 6 h (43-94%), with the activity and yield following the order

[MoO2(L 1 )(MeOH)] >> [MoO2(L 3 )]n ~ [MoO2(L 2 )]n >> [MoO2(L 1 )
]n (Fig. 2). The relevance of the ONS coordination is confirmed by the fact that complex [MoO2(acac)2] gave poorer results while the H2L 1-3 ligands alone were not active at all.

The initial turnover frequencies (TOF20min) for the tested compounds (see SI) are very good with 0.05 Mo % only, the lowest being 645 h -1 for [MoO2(L 1 )]n and the highest one 3360 h -1 for [MoO2(L 1 )(MeOH)]. Under these conditions, the [MoO2(SAP)]2 compound gives a TOF of 453 h -1 with lower selectivity (68%). These results may be compared with those of the organometallic precursor [CpMo(CO)2(η 3 -C3H5)], used at 1% Mo loading, for which a TOF of 97 h -1 was reported under the same conditions (aqueous TBHP), and 310 h -1 using TBHP in decane. [12b] On the other hand, TOFs up to 13000 h -1 were claimed using sugar based ONO ligands. [4] Our results are promising in terms of (i) low Mo loading, (ii) solvent free process (iii) overall performance (activity, selectivity) for "MoO2L" type complexes, (L = tridentate ligand). The [MoO2(L 1 )(MeOH)] system yields a TOF 5 times higher than its corresponding polynuclear compound [MoO2(L 1 )]n. In order to clarify the role of methanol, additional experiments were carried out using different MeOH/[MoO2(L 1 )]n ratios. Excess methanol (m equiv vs. Mo) was added at the beginning of the reaction before the TBHP addition, Exp. Am (m = 0, 1, 12. 5, 25, 50). The kinetic profiles of these experiments are shown in Fig. 3, whereas Fig. 4 compares the initial TOFs and the final conversions. As pointed out earlier for catalytic oxidations with Mo VI complexes coordinated with two bidentate ligands, [4b] the presence of methanol as a solvent should increase the catalytic activity because of the resulting higher polarity of the reaction medium. Experiments A1≤m≤25 indeed show greater activity than experiment A0, but the activity decreases for MeOH amounts greater than 1 equiv. until falling below the level of A0 for a large excess (experiment A50). The quickest and most efficient reaction occurs in case of A1, corresponding to the stoichiometry of the mononuclear methanol adduct. Another interesting experiment (B50) was carried out by running experiment A0 as above, but with the addition of 50 equivalents of MeOH after 90 min from the beginning of the reaction. The kinetic profile of this experiment is shown in Fig. 5, in comparison with those of experiments A0 and A50. As expected, B50 initially follows the same profile as that of the polynuclear catalyst without methanol (A0), and the methanol addition provokes a sudden change of behavior, continuing along the same qualitative profile as experiment A50 after 120 minutes.

These results indicate that, in apparent contrast with the literature suggestions, the solvent effect of the MeOH addition is not acceleration but rather slowdown. A possible interpretation of this effect is to invoke a MeOH dissociation equilibrium from the mononuclear MeOH adduct, liberating a coordination site to yield the catalytically active pentacoordinated [MoO2(L 1 )] species, as indicated in Scheme 3. On the other hand, rationalizing the positive effect of a single MeOH equivalent relative to the polymeric species is less straightforward. It must be noted that both catalysts dissolve completely in the reaction medium at the beginning of the experiment. This suggests that the [MoO2(L 1 )]n complex probably does not retain a polynuclear structure in the catalytic medium. However, it is likely to be present in a dinuclear or oligonuclear soluble form, with the sixth coordination site of the [MoO2(L 1 )] fragment occupied by a dangling hydroxymethyl group from the neighbouring complex. [15] Presumably for entropic reasons, access to the catalytically active 5-coordinate [MoO2(L 1 )] intermediate may be slower from this oligonuclear species relative to the mononuclear MeOH adduct. [16] The fact that no induction time is observed for these experiments suggests that the precatalyst maintains its equilibrium with the catalytic intermediates throughout the process. Indeed, the fact that the two (pre)catalysts (mononuclear MeOH adduct and polymer) with the same ONS ligand display such different catalytic activity suggests that the pre-catalyst also corresponds to the catalyst resting state. We have also investigated the influence of methanol on the catalysis with the polynuclear compounds [MoO2(L 2 )]n (experiments Cm) and [MoO2(L 3 )]n (experiments Dm), with m = 0, 1, 25, 50. In this case, the effect of MeOH addition is much less dramatic, the difference of the various kinetic profiles for the same precatalyst being hardly greater than the experimental error (see Figures S1 andS2, respectively). Since complexes [MoO2(L 2 )]n and [MoO2(L 3 )]n adopt a different structure, as indicated in Scheme 1, with more labile Mo=O•••Mo bridges, it can be assumed that access to the catalytically active pentacoordinated MoO2(L 2 ) and MoO2(L 3 ) species is equally fast from the polynuclear species and from the corresponding mononuclear MeOH adducts. The selectivity patterns for [MoO2(L 2 )]n and [MoO2(L 3 )]n are the same as in the case of [MoO2(L 1 )]nthe selectivity decreases as the amount of added methanol is increased.

A comparison of the activities of the polymeric precatalysts with different ligands (Fig. 6) shows greater activities for those precatalysts where the polymerisation mode involves Mo=O•••Mo bridges (L 2 and L 3 ), [START_REF] Vrdoljak | [END_REF] relative to L 1 which involves Mo(ONS-OH)•••Mo bridges (see Scheme 1). Hence, it seems that the former kind of bridge is more labile than the latter. Further experimental as well as computational investigations of the reaction mechanism are in progress, as well as a broader study of these catalytic systems for the epoxidation of other substrates.

Experimental Section Synthesis

The syntheses and characterizations of monomeric [MoO2(L 1 )(CH3OH)], polymeric complexes [MoO2(L 1-3 )]n had been reported previously. [START_REF] Vrdoljak | [END_REF] [MoO2(SAP)]2 [5,17] and [MoO2(acac)2] [18] were prepared as described in literature. Cyclooctene, aqueous TBHP (70%) and acetophenone were commercially available from Aldrich. 1 H spectra were recorded at 200.1 MHz on a Bruker Advance DPX-200 spectrometer. Catalytic reactions were followed by gas chromatography on an Agilent 6890A chromatograph equipped with FID detector, a DB5-MS capillary column (30 m x 0.32 mm x 0.25 µm). The GC parameters were quantified with authentic samples of the reactants and products. The conversion of cis-cyclooctene and the formation of cyclooctene oxide were calculated from calibration curves (r 2 = 0.999) relatively to acetophenone.

General procedure for the epoxidation of cyclooctene by aqueous TBHP

A mixture of cyclooctene (2.76 mL, 20 mmol), acetophenone (internal reference) and Mo (pre)catalyst (0.01 mmol) was stirred and heated up to 80 °C before addition of aqueous TBHP (70 %, 5.48 mL, 40 mmol). At this point, the mixture is an emulsion, but as the reaction progresses, two phases become clearly visible, a colorless aqueous one and a yellowish organic one. The reaction was followed for 6 h and aliquots were taken on required times. 0.1 mL of organic phase was taken from reaction media and mixed with 2 mL of Et2O. To the solution was added small quantity of MnO2. The mixture was filtered through silica and analyzed by 1 H NMR in CDCl3.

Exp. Am, Cm and Dm -Epoxidation of cyclooctene by the use of [MoO2(L 1-3 )]n -addition of MeOH before t=0 min

The procedure was the same as the previous general procedure except that m equivalents of MeOH per Mo atom were added to reaction mixture together with [MoO2(L 1-3 )]n. The reaction was followed by gas chromatography.

Exp. B50 -Epoxidation of cyclooctene by the use of [MoO2(L 1 )]n -addition of MeOH at t=90 min

The procedure was the same as for Exp. A one, but 50 eq. MeOH were added to the reaction mixture at t=90 min.

Scheme 2 .

 2 Scheme 2. Interconversion of [MoO2(L 1 )(MeOH)] and [MoO2(L 1 )]n by exposure to a different solvent (the grey half-sphere indicates the ONS L 1 ligand).

Figure 1 .Figure 2 .

 12 Figure 1. Kinetic profile of converted cyclooctene vs. time with Mo(VI) (pre)catalysts: [MoO2(L 1 )(MeOH)], [MoO2(L 1 )]n, [MoO2(L 2 )]n, [MoO2(L 3 )]n. Conditions: substrate/(pre)catalyst = 2000:1; T = 80 °C.

Figure 3 .Figure 4 .

 34 Figure 3. Kinetic profile of converted cyclooctene vs. time with [MoO2(L 1 )]n with different MeOH/Mo ratio: from top to bottom (t=360 min): A1 (), A12.5 (), A25 (), A0 (), A50 (∆).

Figure 5 .

 5 Figure 5. Kinetic profile of converted cyclooctene vs. time with [MoO2(L 1 )]n without methanol addition (A0, ------), with methanol addition at t = 0 min (exp. A50, ....  .... ), with methanol addition at t = 90 min (exp. B50, --).

Figure 6 .

 6 Figure 6. Comparative evaluation of TOF20min (h -1 ) for epoxidation with "MoO2(L 1-3 )" (pre)catalysts In conclusion, for the first time, we have shown that Mo VI compounds with a pyridoxal moiety are efficient (pre)catalysts for epoxidation reactions by aqueous TBHP. [MoO2(L 1 )(MeOH)] seemed to be the best (pre)catalyst for the epoxidation of cyclooctene (highest TOF and TON values) in the absence of organic solvents. The comparison between the mononuclear MeOH adducts and the MeOH-free polynuclear material and the effect of the MeOH/Mo ratio for the experiments with added MeOH have revealed a new facet of the solvent effect on the catalytic activity of this family of compounds.Further experimental as well as computational investigations of the reaction mechanism are in progress, as well as a broader study of these catalytic systems for the epoxidation of other substrates.

Acknowledgements

All authors acknowledge CNRS, University Paul Sabatier (Institut Universitaire Paul Sabatier) and Ministry of Science and Technology of the Republic of Croatia (Grant No. 119-1191342-1082) for all research facilities. The fellowship of Jana Pisk was provided by the Ministry of Science and Technology of the Republic of Croatia, the National Foundation of Science and High Education of Croatia (03.01/O-3511-2010) and the French Embassy in Croatia.

Epoxidation