Mathieu Santonja

Stephane Greff

Marie L Le Croller

Olivier P Thomas

Thierry Perez
email: thierry.perez@imbe.fr

Stéphane Greff

Thierry Pérez

Distance interaction between marine cave-dwelling sponges and crustaceans

Keywords: Marine Ecology, Chemical mediation, Porifera, Oxylipins, Mediterranean Sea

à la diffusion de documents scientifiques de niveau recherche, publiés ou non, émanant des établissements d'enseignement et de recherche français ou étrangers, des laboratoires publics ou privés.

INTRODUCTION

Distance interactions regulate the structure and functioning of marine ecosystems and species distribution within a complex mosaic of habitats. There is a need for better understanding of these interactions, in particular when attempting to assess the putative effects of human disturbances on system functioning (van de [START_REF] Van De Koppel | Long-distance interactions regulate the structure and resilience of coastal ecosystems[END_REF]. Chemical mediation plays a key role in such distance interactions by influencing the behavioural ecology of mobile organisms [START_REF] Hay | Marine chemical ecology: chemical signals and cues structure marine populations, communities, and ecosystems[END_REF]. Seawater can also be seen as a chemical "seascape" composed of dissolved and/or adsorbed molecules of diverse origins, a good number of which are released by marine organisms and carry crucial signals for the whole ecosystem [START_REF] Cassier | Olfactory discrimination in juvenile coral reef fishes: response to conspecifics and corals[END_REF].

Surrounded by this complex chemical environment, mobile animals must decipher a complex cocktail of chemical cues, some of which act as kairomones, and adjust their behavioural response by navigating, selecting habitat, locating food or prey, detecting the presence of conspecifics, or defending against competitors and/or predators [START_REF] Heuschele | The chemical ecology of copepods[END_REF]. This complexity also challenges animals' abilities to extract the information required to identify the sources of chemical cues which are crucial for their survival [START_REF] Derby | Neural Processing, Perception, and Behavioral Responses to Natural Chemical Stimuli by Fish and Crustaceans[END_REF].

For example, fishes can detect natural chemical cues at very low concentrations (parts per billion) and, therefore, need to be able to identify the relevant cues from the higher levels of background noise [START_REF] Belanger | Olfactory sensory input increases gill ventilation in male round gobies (Neogobius melanostomus) during exposure to steroids[END_REF].

During the past decades, special attention has been paid to the functioning of tropical coral reefs. One of the greatest challenges has been to better understand the life cycles of littoral populations of marine species living in fragmented habitats in a vast and complex ocean matrix, species which may have dispersal and larval development phases offshore, followed by the recruitment of juveniles on randomly distributed reefs [START_REF] Myrberg | (ed) Coral reef fishes: dynamics and diversity in a complex ecosystem[END_REF]. Several pioneer studies demonstrated that marine fish and crustacean larvae could distinguish between oceanic and reef waters [START_REF] Atema | Larval reef fish could use odour for detection, retention and orientation to reefs[END_REF][START_REF] Lecchini | Larval sensory abilities and mechanisms of habitat selection of a coral reef fish during settlement[END_REF][START_REF] Gerlach | Smelling home can prevent dispersal of reef fish larvae[END_REF][START_REF] Dixson | Coral reef fish smell leaves to find island homes[END_REF]). These larvae were shown to be capable of selecting a given habitat among the great variety of substrates found in tropical waters [START_REF] Huijbers | Post-larval French grunts (Haemulon flavolineatum) distinguish between seagrass, mangrove and coral reef water: Implications for recognition of potential nursery habitats[END_REF][START_REF] Munday | Ocean acidification impairs olfactory discrimination and homing ability of a marine fish[END_REF][START_REF] Lecchini | Ecological determinants and sensory mechanisms in habitat selection of crustacean postlarvae[END_REF][START_REF] Devine | Homing ability of adult cardinalfish is affected by elevated carbon dioxide[END_REF][START_REF] Igulu | Orientation from open water to settlement habitats by coral reef fish: behavioral flexibility in the use of multiple reliable cues[END_REF]. For example, [START_REF] Huijbers | Post-larval French grunts (Haemulon flavolineatum) distinguish between seagrass, mangrove and coral reef water: Implications for recognition of potential nursery habitats[END_REF] reported that fish recruits discriminate chemical cues from mangroves, seagrass beds and coral reefs and that a given reef fish actively prefers the odour of its home coral reef. [START_REF] Lecchini | Transmission distance of chemical cues from coral habitats: implications for marine larval settlement in context of reef degradation[END_REF] also demonstrated that fish, crustacean and cephalopod larvae were significantly more attracted by chemical cues from living corals than by dead colonies. Further studies have highlighted how environmental disturbances leading to coral reef destructions, such as ocean acidification [START_REF] Munday | Ocean acidification impairs olfactory discrimination and homing ability of a marine fish[END_REF][START_REF] Devine | Homing ability of adult cardinalfish is affected by elevated carbon dioxide[END_REF][START_REF] Leduc | Effects of acidification on olfactory-mediated behaviour in freshwater and marine ecosystems: a synthesis[END_REF], seawater warming [START_REF] Mccormick | Influence of habitat degradation on fish replenishment[END_REF] or pollution [START_REF] O'connor | Sediment pollution impacts sensory ability and performance of settling coral-reef fish[END_REF], may affect marine chemical mediation.

Additionally, these changes in abiotic conditions may also alter the organism's metabolism and, thus, its ability to produce specialized metabolites and may consequently impact chemical mediation between organisms [START_REF] Fisher | Alteration of the chemical environment disrupts communication in a freshwater fish[END_REF][START_REF] Munday | Ocean acidification impairs olfactory discrimination and homing ability of a marine fish[END_REF][START_REF] Leduc | Effects of acidification on olfactory-mediated behaviour in freshwater and marine ecosystems: a synthesis[END_REF]. Surprisingly, even though chemicals are involved in these biotic interactions and in overall ecosystem functioning, few studies have attempted to identify the chemical nature of distance marine chemical cues [START_REF] Lecchini | Larval sensory abilities and mechanisms of habitat selection of a coral reef fish during settlement[END_REF][START_REF] Lecchini | Ecological determinants and sensory mechanisms in habitat selection of crustacean postlarvae[END_REF][START_REF] Lecchini | Transmission distance of chemical cues from coral habitats: implications for marine larval settlement in context of reef degradation[END_REF][START_REF] Rasher | Marine and terrestrial herbivores display convergent chemical ecology despite 400 million years of independent evolution[END_REF].

In comparison to this knowledge gained in tropical reefs, chemical mediation in temperate benthic ecosystems has been poorly studied. Sponges can dominate in terms of biomass and diversity in numerous benthic ecosystems, both in tropical and temperate regions [START_REF] Diaz | Sponges: an essential component of Caribbean coral reefs[END_REF][START_REF] Gerovasileiou | Marine caves of the Mediterranean sea: a sponge biodiversity reservoir within a biodiversity hotspot[END_REF][START_REF] Wulff | Ecological Interactions and the Distribution, Abundance, and Diversity of Sponges[END_REF]. They are also well known for their ability to produce a great chemical diversity (e.g. [START_REF] Paul | Marine chemical ecology in benthic environments[END_REF]. However, there is a lack of knowledge concerning the ecological functions of these chemical compounds which can be released into the surrounding water [START_REF] Thompson | Exudation of biologically-active metabolites in the sponge Aplysina fistularis. I. Biological evidence[END_REF][START_REF] Walker | Exudation of biologically-active metabolites in the sponge Aplysina fistularis. II. Chemical evidence[END_REF][START_REF] Ternon | Spherulization as a process for the exudation of chemical cues by the encrusting sponge C. crambe[END_REF]. Today, sponge chemical ecology is mainly based on the assessment of repulsive properties, the so-called "chemical defences", against predators or competitors for space [START_REF] Paul | Marine chemical ecology in benthic environments[END_REF][START_REF] Pawlik | The chemical ecology of sponges on Caribbean reefs: natural products shape natural systems[END_REF]. Even though sponges are often considered to be dominant components of benthic ecosystems, the putative role of the portion of their specialized metabolites released into the water column remains unclear and few studies have demonstrated a positive influence of these cues on the recruitment of invertebrate larvae (e.g. [START_REF] Bingham | Influence of sponges on invertebrate recruitment: a field test of allelopathy[END_REF]. We hypothesize that the great chemical diversity produced by sponges may construct a true chemical seascape which may be the basis of a still underestimated network of interactions shaping biodiversity.

We surmise that underwater caves constitute good ecosystem models in which to test this hypothesis. Sponges are predominant from the semi-dark zone where they coexist with cnidarians and bryozoans to the darkest parts of the cave where they can represent 100 % of the sessile biodiversity [START_REF] Gerovasileiou | Marine caves of the Mediterranean sea: a sponge biodiversity reservoir within a biodiversity hotspot[END_REF]. In low or no light conditions, other putative producers of chemicals such as macroalgae [START_REF] Amsler | Algal Chemical Ecology[END_REF] are absent or are restricted to the entrance of this habitat. Experimenting in underwater caves also avoids, or at least attenuates, variations of abiotic factors present in the open sea such as hydrodynamics, nutrient and resource bioavailability and, in some cases, seawater temperature [START_REF] Rastorgueff | Feeding strategies and resource partitioning mitigate the effects of oligotrophy for marine cave mysids[END_REF].

Underwater caves can thus be considered as in situ mesocosms where many ecological processes and interactions are simplified. Finally, considering their very low resilience facing major environmental threats [START_REF] Gunn | The invertebrate community of the Peak-Speedwell cave system, Derbyshire, England -pressures and considerations for conservation management[END_REF], a better understanding of this ecosystem functioning is needed to propose suitable management and conservation plans [START_REF] Gunn | The invertebrate community of the Peak-Speedwell cave system, Derbyshire, England -pressures and considerations for conservation management[END_REF][START_REF] Bussotti | Spatial and temporal variability of sessile benthos in shallow Mediterranean marine caves[END_REF][START_REF] Gerovasileiou | Marine caves of the Mediterranean sea: a sponge biodiversity reservoir within a biodiversity hotspot[END_REF]Rastorgueff et al. 2015).

This study focuses on two dominant components of cave ecosystem functioning: sponges, which are active suspension feeders and which compose the vast majority of the sessile fauna, and swimming crustaceans which act on bentho-pelagic couplings through their migration in and out of caves (Rastorgueff et al. 2015). Our aim was to assess the influence of the chemical seascape of a cave which is most likely primarily determined by sponges on the swimming behaviour of crustaceans (i.e. attraction or repulsion). We combined the use of (i) an experimental device to assess the behavioural response of the crustaceans, and (ii) a metabolomics fingerprinting approach to decipher the chemical composition of the various seawater treatments. To conduct our experiments, we selected representatives of the sponge community found near the entrance in semi-dark conditions of Mediterranean underwater caves, and four crustacean species with contrasting ecological habits: two cave-dwelling crustaceans and two crustaceans inhabiting shallow water environments.

MATERIAL AND METHODS

Study location and species

This study was conducted using the aquaria facilities of the Marine Station of Endoume, Marseille, France. Four characteristic sponge species of the semi-dark community (Aplysina cavernicola (Vacelet, 1959), Haliclona fulva (Topsent, 1893), Oscarella tuberculata (Schmidt, 1868) and Spongia officinalis (Linnaeus, 1759); Supplementary Fig. S1) were collected in a cave in the Riou Archipelago at an average of 20 m depth (43°12'N; 5°22'E) and were maintained alive in aquaria. These species were selected because part of their specialized metabolome had already been described [START_REF] Garrido | New furanoterpenoids from the sponge Spongia officinalis[END_REF][START_REF] Ivanisevic | Metabolic fingerprinting as an indicator of biodiversity: towards understanding inter-specific relationships among Homoscleromorpha sponges[END_REF][START_REF] Manzo | Bioactive terpenes from Spongia officinalis[END_REF][START_REF] Nuzzo | Fulvynes, antimicrobial polyoxygenated acetylenes from the Mediterranean sponge Haliclona fulva[END_REF][START_REF] Reverter | Secondary Metabolome Variability and Inducible Chemical Defenses in the Mediterranean Sponge Aplysina cavernicola[END_REF]personal unpublished data). In the same cave, we also collected 70 L of seawater to act as a positive control in our first experiment.

The four crustacean species selected to perform the chemosensory assays were two mysid species (Hemimysis margalefi Alcaraz, Riera and Gili, 1986 and Leptomysis sp.) and two palaemonid species (Palaemon serratus (Pennant, 1777) and Palaemon elegans Rathke, 1837) (Supplementary Fig. S2). Hemimysis margalefi (mean length ± SD, 2.7 ± 0.1 mm) was collected in the same cave as the sponges, where it usually forms dense swarms. This cave-dwelling mysid undertakes circadian migrations in order to find food outside the cave at night or early morning [START_REF] Rastorgueff | Feeding strategies and resource partitioning mitigate the effects of oligotrophy for marine cave mysids[END_REF]), and we hypothesized that its return towards the cave may be influenced by a chemical seascape composed primarily of sponge chemical cues. Leptomysis sp. (8.3 ± 0.3 mm) was collected in shallow waters (8 m depth) adjacent to the Marine Station aquarium seawater pumping site (43°16.80'N, 5°20.95'E). This benthic community is dominated by macrophytes such as Posidonia oceanica and is, thus, a totally different chemical seascape which is devoid of any sponge influence. Palaemon serratus (13.8 ± 0.3 mm) and P. elegans (8.2 ± 0.1 mm) individuals were offspring of gravid females that were captured in the Mediterranean Sea and had been raised in aquaria, so were totally naïve regarding the experimental conditions. Palaemon serratus is known to live in semi-dark to dark environments, and we hypothesized that its occurrence in caves may be related to the chemical seascape, whereas P. elegans lives in very shallow waters among macroalgae assemblages, so they should not be influenced by a seascape conditioned by sponges. For the duration of their time spent in aquaria, all four crustacean species were fed every 2 days with Artemia salina nauplii.

Chemosensory trials

A Y-shaped glass flume providing a choice of two channels was set up according to a protocol developed by [START_REF] Gerlach | Smelling home can prevent dispersal of reef fish larvae[END_REF] to assess the crustacean behavioural response when faced with the choice of different seawater treatments: open flow seawater as the control seawater (CS); cave seawater (+CS), considered here as a positive control; and two types of conditioned seawater, sponge conditioned seawater (SCS) and sponge chemical extracts dissolved in seawater (SCE). Two 10 L tanks were connected to the flume by pipes, creating a constant gravity-driven flow of 50 mL min -1 per upstream channel. Prior to the experiments, both flows were measured by adding fluorescein to one tank to ensure that the flows were similar and mixed only in the downstream compartment. The crustaceans were introduced at the downstream end of the apparatus, where they were free to remain or to select and swim towards one of the two channels connected to the tanks (Supplementary Fig. S3). Each assay was performed with a single specimen which was maintained for a 1-min acclimation period followed by a 5-min testing period. During the selection phase of the test, the position of the crustacean in the downstream compartment or in the right or left channels was regularly recorded at 5-sec intervals. After each test, the flume chamber was emptied and washed with the CS. The seawater treatments were then switched from one channel to the other, providing a control for potential channel preferences that were not associated with the water source. After seawater switching, the entire assay was repeated with a new specimen, with each individual being used in a single assay. In this way, the time (in seconds) spent by the crustacean individual in each of the three compartments of the choice flume (i.e. the downstream and the two channels) was recorded.

Using the protocol described above, three experiments were conducted with a minimum of 40 individuals per experiment and species, with the exception of P. elegans, for which only 25 individuals were tested per experiment. Moreover, these experiments were conducted both in the morning (half of the total individuals; 7 h -11 h UTC) and in the afternoon (half of the total individuals; 14 h -18 h UTC) in order to assess the daily variability of the crustaceans' response to the seawater treatments. For each experiment, +CS and the two types of conditioned seawater (SCS and SCE) were tested against CS. Because palaemonids were not available for the first experiment (CS vs. +CS), only the two mysids were used. All four crustaceans were used for the second (CS vs. SCS) and the third (CS vs. SCE) experiments.

The SCS was expected to contain the sponge community exometabolome (i.e. the metabolites naturally released into the seawater by the four selected sponge species). The SCS was obtained by maintaining living individuals of the four sponge species, measuring about 25 cm 3 each, in a 10 L aerated-tank for 16 hours. The SCE was expected to contain the global metabolome of the same sponge community, that is to say, all metabolites contained in the tissues of the four selected sponges. This SCE was obtained from the sponges used to obtain the SCS, which were weighed, frozen, ground in a blender, filtered through a 500 µm sieve and then mixed with seawater in order to obtain a homogenous solution. The sponge community used to prepare the SCS and SCE represented a total weight of 108 ± 10 g.

To ensure that the modifications of the seawater used to prepare the SCS and SCE was due to the introduction of sponge exometabolomes or extracts only and not by a microbial community present in the pumped open flow seawater, a further CS was prepared for the metabolomic analyses by maintaining this open flow seawater for 16 hours in the same condition as the SCS.

Metabolomic analyses

An untargeted metabolomic approach was used to compare the metabolite composition of the different seawater treatments: the control seawater (CS), the cave seawater (+CS) and the two conditioned seawater treatments (SCS and SCE). . Briefly, a volume of 500 mL of seawater samples was collected in the tanks during the two-flume choice experiments and passed successively through 0.45 µm pore-sized nylon filters and octadecyl-bonded silica extraction discs (Empore TM C18 SPE discs) to obtain particulate and dissolved phases of each seawater. The filters were extracted for particulate phase (or eluted for dissolved phase) with methanol. The extracts were then concentrated to dryness, treated to remove salts, dissolved in methanol and filtered at 0.2 µm before metabolomic analysis. On-line Ultra High-Performance Liquid Chromatography coupled to a High-Resolution Mass Spectrometer (UHPLCHRMS) analyses were performed using a Dionex Ultimate 3000 system equipped with an autosampler and connected to a quadrupole Time of Flight (qToF) mass spectrometer equipped with an electrospray ionization interface (Bruker Impact II) to record mass spectra in the positive and negative modes (see Supplementary Information for detailed methods).

Following the UHPLC-HRMS data acquisition, base peak chromatograms (BPC) were exported as line spectra and converted into the netCDF file format to process unit mass resolution data in centroid mode with XCMS [START_REF] Smith | XCMS: Processing mass spectrometry data for metabolite profiling using nonlinear peak alignment, matching, and identification[END_REF]) on R software (3.4.2). The XCMS approach involved peak picking and integration, peak grouping for identification between samples, chromatogram alignment to avoid retention time deviation and, finally, peak filling to avoid missing values in chromatograms. XCMS generated a matrix (ions/retention time × sample) that was exported on Microsoft Excel. Home-made scripts were used to clean the matrix, removing 1) ions detected in solvent blanks (ratio pools/blanks < 10),

2) ions presenting an intensity variation above 30% for pool samples, and 3) ions with correlated intensities for pool samples (threshold = 0.8). Extracted Ion Chromatograms of variable importance in projection (VIP) chemomarkers were drawn to measure their concentrations in the different seawater treatments, taking into account the most important value of ion intensity at each VIP retention time. The heatmap based on data acquired in positive mode was finally selected to tentatively annotate chemomarkers of seawater samples using the molecular formulae provided by Bruker Data-analysis 4.2.

Statistical analyses

Paired samples t tests (or Wilcoxon tests when normality and homoscedasticity conditions were not met) were performed to compare the time spent in each of the two upstream channels containing the different seawater treatments (CS vs. +CS; CS vs. SCS; CS vs. SCE), in order to determine the channel chosen by each crustacean species. Two-sample t tests (or Mann Whitney tests when normality and homoscedasticity conditions were not met) were performed to compare the time spent in the channels filled with CS, +CS, SCS or SCE between morning and afternoon, in order to determine if crustacean choice is affected by the time of day.

Principal Component Analyses (PCA) were performed to explore and identify similarities between the chemical fingerprints of the different seawater samples. A permutation test (999 permutations, PPLS-DA models, based on a double cross-validation, outer loop = 4 fold crossvalidation, and inner loop = 3 fold cross-validation) was performed for the differentiation of the chemical fingerprints of the different seawater samples followed by post hoc permutational pairwise test (Hervé, 2018). The classification error rate (CER, 0 -1) was calculated to estimate the strength of the generated models. Kruskal Wallis tests, followed by a post-hoc multiple range test (Fisher's Least Significant Difference), were performed to test differences in chemomarker concentrations between seawater treatments.

Multivariate analyses (PCA) and heatmaps were performed with MetaboAnalyst 3.5 [START_REF] Xia | Using MetaboAnalyst 3.0 for Comprehensive Metabolomics Data Analysis[END_REF]. Univariate analyses were performed with R software (version 3.4.2.) and Statgraphics centurion (version 15.2.11.0).

RESULTS

Control experiment

When both upstream channels were filled with CS, the four crustaceans showed no significant preference between upstream channels (Supplementary Fig. S4) and remained mainly in the downstream compartment (Supplementary Fig. S5). These results confirmed the equivalence of the two channels for the assay. Moreover, both mysid species exhibited higher locomotor activity than the two palaemonid species, staying twice as long in the upstream channels (Supplementary Fig. S4).

Comparative crustacean responses to the seawater treatments

During the 5-min tests of +CS vs. CS, most crustaceans exhibited stationary swimming behaviour at the intersection of the seawater currents from the two upstream channels before exhibiting their preference. However, occasionally after making their first choice, individuals would switch and explore the opposite channel, showing constant activity while investigating the seawater from both upstream channels. Hemimysis margalefi showed a clear preference for the +CS, staying 16 times longer in this channel (Fig. 1) than in the CS. In contrast, Leptomysis sp. demonstrated a significant preference for the CS channel, staying 4 times longer in this channel (Fig. 1) than in the +CS. The behaviour of the two mysids varied with the time of day, with H. margalefi having a higher preference for the +CS in the morning (Fig. 2). Interestingly, Leptomysis sp. had a higher preference for the CS in the afternoon (Fig. 2).

In the SCS vs. CS experiment, Hemimysis margalefi exhibited a significant preference for the SCS, staying 4 times longer in this channel (Fig. 1) than in the CS. This behaviour varied with the time of day, with a higher preference for the SCS in the morning, when it spent 7 times longer than in the CS channel, whereas this difference was only 2.5 times longer in the afternoon (Fig. 3). Palaemon serratus also exhibited a preference for the SCS compared to the CS channel (Fig. 1), but this attraction was less pronounced than for H. margalefi and was not affected by the time of day (Fig. 3). Comparatively, Leptomysis sp. confirmed its clear preference for the CS channel (Fig. 1) and, in this experiment too, their behaviour varied with the time of day, with no clear preference in the morning, but with a 5-fold longer time spent in the CS channel than in the SCS in the afternoon (Fig. 3). Finally, as was the case for Leptomysis sp., P. elegans showed no preference in the morning but stayed 4 times longer in the CS than in the SCS channel in the afternoon (Fig. 3).

In the SCE vs. CS experiment, both H. margalefi and P. serratus clearly avoided the SCE channel and spent about 15 times longer in the CS channel compared to the SCE channel (Fig. 1). This trend was less marked for Leptomysis sp. and P. elegans, as these crustaceans stayed in the CS channel only 2 to 3 times longer than they did in the SCE channel (Fig. 1). The two cave-dwelling crustaceans (i.e. H. margalefi and P. serratus) presented the same behavioural response regardless of the time of day, whereas Leptomysis sp. and P. elegans exhibited a significant avoidance of SCE in the afternoon only (Fig. 4). Moreover, the swimming behaviour appeared quite different than in the previous experiments. Hemimysis margalefi and P. serratus did not exhibit a stationary phase at the intersection of the seawater currents, but they swam more quickly and presented a faster choice, mostly in favour of the CS channel. In a distinct manner, Leptomysis sp. showed a reduced swimming activity and spent most of its time on the bottom of the downstream compartment (Supplementary Fig. S5).

Metabolomic analyses

The comparative untargeted analyses of the CS, +CS, SCS and SCE chemical fingerprints were performed by UHPLC-HRMS in both negative and positive modes and for both dissolved and particulate phases. After XCMS processing and filtering, 156 ions in the negative mode and 144 ions were detected in the positive mode, and these were used to compare the chemical fingerprints of the various seawater treatments (Fig. 5, Fig. S9). As a preliminary result, no change could be detected when comparing the chemical fingerprints of the two CS, i.e. the CS freshly pumped to perform the behavioural experiment or the CS kept for 16 hours as was the conditioned seawater SCS.

The chemical fingerprints of the dissolved phases obtained in both negative and positive modes resulted in the separation of +CS on one side and CS, SCS and SCE on the other side along the first axis of the PCA (Fig. 5, Table S1). Along the second axis, the chemical fingerprints recorded in the negative mode resulted in the grouping of SCS and SCE that overlapped opposite to CS (Fig. 5), whereas in the positive mode, SCS grouped with CS opposite to SCE (Fig. 5), but SCS and CS appeared different according to the post hoc permutational pairwise test (Table S1). Chemical fingerprints of particulate phases obtained in both negative and positive modes showed similar patterns with the grouping of CS with SCS separated from both SCE and +CS (Fig. 5).

To highlight some chemical markers potentially responsible for the crustaceans' behaviour, heatmaps of the most important features that discriminate groups in both modes and phases were constructed (Fig. 6, and Supplementary Figs. S6, S7 and S8). Many features present in SCE waters in high quantities may help to explain the crustaceans' repulsion. Among them, the brominated derivatives known from A. cavernicola were easily identifiable on the basis of their isotopic pattern, exact mass and in comparison with standards (Fig. S10). For example, aeroplysinin-1 (m/z 335.8871, rt 400 s, M338T401 on Fig. S7) was identified mostly in the dissolved phase and aerothionin (m/z 812.8406, rt 470 s, M819T471 and M820T472 on Fig. S8, and possible stereoisomer with the same isotopic pattern at rt 500: M817T501 and M819T502, Figs. S8 andS10) was identified mostly in the particulate phase. On the contrary, only a few chemical features were found to possibly explain the attraction of H. margalefi by both +CS and SCS. The chemical fingerprints obtained in the negative mode from the dissolved phases were the only ones separating CS from +CS and SCS/SCE (Fig. 5).

In the positive mode of the dissolved phase, two sets of features were tentatively annotated. The 1 st set of features grouped ions present in both +CS and SCE (Supplementary Table S2, Figs. 6 and7). Four metabolites, A (C24H44N4O4), B (C30H55N5O5), C (C36H66N6O6) and D (C42H77N7O7), were detected in this group and are likely small peptides made of leucine or iso-leucine units. Their concentrations were significantly higher in +CS, whereas the few visible differences between SCS and CS were never statistically significant (Fig. 7). The 2 nd set of features grouped ions which were more concentrated in SCS and SCE, present in +CS and overall poorly represented in CS (Supplementary Table S2, Figs. 6 and7). They were consistently significantly more concentrated in SCS than in other waters. However, although some of them were twice as concentrated in +CS than in CS, these differences were generally not supported statistically except for the metabolite L (C17H34N2O3) detected in higher concentration in SCS and +CS compared to CS (Fig. 7). Molecular formulae of the marker metabolites E (C18H36O6), F (C18H30O3), G (C18H34O5) and H (C14H26O3) were confidently assigned thanks to the high accuracy of the mass and the consistency of the molecular formulae provided by data analysis. These compounds likely belong to the oxylipin family, some derivatives of a family previously found in the marine environment [START_REF] Qiao | Steroids and an oxylipin from an algicolous isolate of Aspergillus flavus[END_REF][START_REF] Lamari | Specificity of lipoxygenase pathways supports species delineation in the marine diatom genus Pseudo-nitzschia[END_REF][START_REF] Lauritano | New oxylipins produced at the end of a diatom bloom and their effects on copepod reproductive success and gene expression levels[END_REF]. Halogenated metabolites harbouring the same number of carbons were also detected, I (C18H31BrN2O5), J (C18H34Cl2O4) and K (C18H33ClO4).

DISCUSSION

Distance interactions due to the putative role of chemical mediation in habitat detection by fishes and crustaceans have been previously studied among benthic ecosystems dominated by macroalgae [START_REF] Lecchini | Ontogenetic changes in habitat selection during settlement in a coral reef fish ecological determinants and sensory mechanisms[END_REF][START_REF] Lecchini | Ecological determinants and sensory mechanisms in habitat selection of crustacean postlarvae[END_REF][START_REF] Lecchini | Fish larvae prefer coral over algal water cues: implications of coral reef degradation[END_REF][START_REF] Lecchini | Visual and chemical cues in habitat selection of sepioid larvae[END_REF] or seagrasses [START_REF] Nagelkerken | How important are mangroves and seagrass beds for coral-reef fish? The nursery hypothesis tested on an island scale[END_REF][START_REF] Arvedlund | The importance of chemical environmental cues for juvenile Lethrinus nebulosus Forsskal (Lethrinidae, Teleostei) when settling into their first benthic habitat[END_REF][START_REF] Huijbers | Post-larval French grunts (Haemulon flavolineatum) distinguish between seagrass, mangrove and coral reef water: Implications for recognition of potential nursery habitats[END_REF][START_REF] Igulu | Orientation from open water to settlement habitats by coral reef fish: behavioral flexibility in the use of multiple reliable cues[END_REF], in coral reefs [START_REF] Lecchini | Larval sensory abilities and mechanisms of habitat selection of a coral reef fish during settlement[END_REF][START_REF] Lecchini | Ontogenetic changes in habitat selection during settlement in a coral reef fish ecological determinants and sensory mechanisms[END_REF][START_REF] Huijbers | Post-larval French grunts (Haemulon flavolineatum) distinguish between seagrass, mangrove and coral reef water: Implications for recognition of potential nursery habitats[END_REF][START_REF] Ben-Tzvi | Role of coral-derived chemical cues in microhabitat selection by settling Chromis viridis[END_REF][START_REF] Lecchini | Ecological determinants and sensory mechanisms in habitat selection of crustacean postlarvae[END_REF][START_REF] Mccormick | Influence of habitat degradation on fish replenishment[END_REF][START_REF] Lecchini | Visual and chemical cues in habitat selection of sepioid larvae[END_REF][START_REF] Devine | Homing ability of adult cardinalfish is affected by elevated carbon dioxide[END_REF][START_REF] Cassier | Olfactory discrimination in juvenile coral reef fishes: response to conspecifics and corals[END_REF][START_REF] Igulu | Orientation from open water to settlement habitats by coral reef fish: behavioral flexibility in the use of multiple reliable cues[END_REF][START_REF] Lecchini | Fish larvae prefer coral over algal water cues: implications of coral reef degradation[END_REF][START_REF] Lecchini | Transmission distance of chemical cues from coral habitats: implications for marine larval settlement in context of reef degradation[END_REF]) and in mangroves [START_REF] Huijbers | Post-larval French grunts (Haemulon flavolineatum) distinguish between seagrass, mangrove and coral reef water: Implications for recognition of potential nursery habitats[END_REF][START_REF] Igulu | Orientation from open water to settlement habitats by coral reef fish: behavioral flexibility in the use of multiple reliable cues[END_REF]). For some model interactions, such as clown fishes and sea anemones, their description demonstrated how these processes are crucial for ecosystem functioning, and how this mediation can be affected by global change [START_REF] Arvedlund | Do the anemonefish Amphiprion ocellaris (Pisces: Pomacentridae) imprint themselves to their host sea anemone Heteractis magnifica (Anthozoa: Actinidae)?[END_REF][START_REF] Arvedlund | Host recognition and possible imprinting in the anemonefish Amphiprion melanopus (Pisces: Pomacentridae)[END_REF][START_REF] Dixson | Coral reef fish smell leaves to find island homes[END_REF][START_REF] Munday | Ocean acidification impairs olfactory discrimination and homing ability of a marine fish[END_REF][START_REF] Dixson | Predation risk assessment by larval reef fishes during settlement-site selection[END_REF][START_REF] Dixson | Chemically mediated behavior of recruiting corals and fishes: a tipping point that may limit reef recovery[END_REF][START_REF] Lecchini | Transmission distance of chemical cues from coral habitats: implications for marine larval settlement in context of reef degradation[END_REF]).

Here, we report for the first time clear behaviours of cave-dwelling crustaceans attracted by cave seawater (+CS) and sponge conditioned seawater (SCS), and the concomitant presence of chemomarkers in these waters. Sponges being dominant in benthic communities such as underwater caves, we hypothesized that sponges and/or their associated microbiota may be responsible for the production of chemical cues that significantly contribute to the network of chemical interactions shaping the ecosystem. Conversely to previous studies in this field, we anticipated that only part of the chemical diversity produced by these sessile invertebrates would contribute to an overall chemical sea-scape conditioning the network of interactions.

Indeed, most previous studies on sponge chemical ecology used tissue homogenates or crude extracts (i.e. the global metabolome) to highlight their deterrent properties against predators, competitors for space or microorganisms [START_REF] Bakus | Toxicity in sponges and holothurians: a geographic pattern[END_REF][START_REF] Thompson | Screening and bioassays for biologically-active substances from forty marine sponge species from San Diego, California, USA[END_REF][START_REF] Thacker | Allelopathic interactions between sponges on a tropical reef[END_REF][START_REF] Kubanek | Multiple defensive roles for triterpene glycosides from two Caribbean sponges[END_REF][START_REF] Pawlik | Does the odor from sponges of the genus Ircinia protect them from fish predators?[END_REF][START_REF] Pawlik | The chemical ecology of sponges on Caribbean reefs: natural products shape natural systems[END_REF]. Our experiment exposing crustaceans to SCE confirmed the strong deterrent effect of seawater containing the entire sponge metabolome. Most likely, deterrent or toxic properties of specialized metabolites present in the sponges are responsible for these behavioural responses. In line with this hypothesis, aerothionin, which is one of the discriminating compounds of the SCE, and other compounds of the same family had already been identified as feeding deterrents in other bioassays and may be responsible for the observed repulsive effects [START_REF] Thoms | Chemical defense of Mediterranean sponges Aplysina cavernicola and Aplysina aerophoba[END_REF][START_REF] Gochfeld | Disease and nutrient enrichment as potential stressors on the Caribbean sponge Aplysina cauliformis and its bacterial symbionts[END_REF]. Interestingly, none of the main specialized metabolites previous known in the four sponges tested were detected in the SCS or the +CS, so they do not appear to belong to what can be called the sponge exometabolome and, thus, do not contribute to the chemical seascape. So far, the exudation of major compounds has been demonstrated in the single case of the sponge Crambe crambe [START_REF] Ternon | Spherulization as a process for the exudation of chemical cues by the encrusting sponge C. crambe[END_REF]), but there is crucial need for new experiments to better distinguish between the global metabolome, which is usually extracted from organisms using solvent, and the exometabolome which is the part naturally released by an organism into the surrounding water.

Following previous hypotheses by [START_REF] Bingham | Influence of sponges on invertebrate recruitment: a field test of allelopathy[END_REF], our study demonstrates for the first time that seawater conditioned by a sponge assemblage (SCS), potentially containing components of the exometabolome only, exerts an attractant effect on some mobile invertebrate species, leading to a specific behavioural response related to their ecology. Demonstrating such a "positive" effect of sponge compounds would have been impossible with only the rather traditional use of crude extracts or pure major compounds in our experimental plan. This past strategy explains the important knowledge gap that exists concerning processes of chemical mediation within marine benthic ecosystems, in particular where ecosystems are dominated by sponges. Few studies have attempted to demonstrate the putative release of metabolites into the surrounding medium [START_REF] Walker | Exudation of biologically-active metabolites in the sponge Aplysina fistularis. II. Chemical evidence[END_REF]Porter and Targett 1988;[START_REF] Duque | Exudation of low molecular weight compounds (thiobismethane, methyl isocyanide, and methyl isothiocyanate) as a possible chemical defense mechanism in the marine sponge Ircinia felix[END_REF][START_REF] Ternon | Spherulization as a process for the exudation of chemical cues by the encrusting sponge C. crambe[END_REF]. After characterizing the exudation of chemical cues by the sponge Crambe crambe, and assessing the toxic activity of these compounds on invertebrate larvae, [START_REF] Ternon | Spherulization as a process for the exudation of chemical cues by the encrusting sponge C. crambe[END_REF] hypothesized that this process might explain the ecological success of this encrusting sponge. Here, we provide the first demonstration that some sponge metabolites may also be involved in distance interactions, acting as kairomones to shape the behaviour of mobile organisms and, thus, structuring biodiversity. We thus confirm the hypothesis of [START_REF] Bingham | Influence of sponges on invertebrate recruitment: a field test of allelopathy[END_REF] that chemicals released by sponges have the potential to attract organisms. Moreover, we have demonstrated a species-specific response to conditioned seawater, since H. margalefi and P. serratus only showed a significant attraction for the seawater containing the exometabolome of the sponge community. The opposite response of Leptomysis sp. can be explained by the fact that the CS was pumped from heart to the Leptomysis sp. Habitat, which is dominated by macrophytes and macroalgae and where sponges are poorly distributed.

The responses of our cave-dwelling species support our hypothesis that chemical cues released by the sponges located at the entrance of underwater caves might be a key factor in the homing behaviour of H. margalefi and a refuge signal for P. serratus. The difference recorded between their behavioural responses is likely related to their life habits. Hemimysis margalefi is a cave-dweller living in swarms and is thought to migrate out of the cave for night feeding [START_REF] Rastorgueff | Feeding strategies and resource partitioning mitigate the effects of oligotrophy for marine cave mysids[END_REF]Rastorgueff et al. 2015). This circadian movement is presumed to be linked to the decrease in light intensity [START_REF] Passelaigue | Distribution and circadian migrations of the cavernicolous mysid Hemimysis speluncola Ledoyer[END_REF], at least to explain how these mysids get out of the cave at the end of the day. However, our goal was to provide clues to support the idea that the return to the cave in the night or early morning may be related to the chemical seascape of the cave, through chemical cues potentially produced by sponges guiding these mysids to the entrance of their habitat. We acknowledge that we only conducted our experiments in the morning, and future experiments could be performed in darkness to mimic if the same trend is observed during the night. Interestingly, H. margalefi exhibited the same positive attraction when faced with the choice of the natural cave seawater and the sponge conditioned seawater containing exometabolomes. A better understanding of these migrations of cave-dwelling organisms is crucial as they represent important vectors of organic matter transfer from the outside euphotic littoral zone to various locations inside caves, where this matter becomes available to other cave dwellers such as molluscs, crustaceans and teleost fishes [START_REF] Rastorgueff | Feeding strategies and resource partitioning mitigate the effects of oligotrophy for marine cave mysids[END_REF]. There is likely a strong link between this type of migration pattern, and how it mitigates cave oligotrophy, and the diversity of sessile organisms at the origin of the chemical seascape.

Palaemon serratus is a crustacean which may occasionally inhabit underwater caves. In this case too, we hypothesize that the sponge seascape might influence the swimming behaviour of this species and contribute to locate its habitat refuge. Interestingly, P. serratus spent most of its time in the downstream compartment and exhibited a much less pronounced attraction for the sponge conditioned seawater, compared to H. margalefi. In the case of palaemonids, the process of chemical mediation in habitat detection might not be related to gradients of waterborne molecules only. Whereas mysids are swimming crustaceans, palaemonids swim close to and walk on the bottom using their well-developed legs. They continuously explore the bottom, sensing more hydrophobic molecules with leg chemoreceptors. They also use pincers to bring fragments of substrate into contact with chemosensory mouthparts in order to follow hydrophobic cues adherent to the substrate [START_REF] Mollo | Sensing marine biomolecules: smell, taste, and the evolutionary transition from aquatic to terrestrial life[END_REF].

Variations of the behavioural responses with time of day differed among the species tested. The cave-dwelling mysid seemed to be much more attracted by sponge cues in the mornings, whereas the palaemonid showed no variation, which tends to confirm that their response does not rely on the same ecological process. Daily rhythm of chemosensitivity has already been reported for several arthropods, such as the Tsetse fly Glossina morsitans morsitans which presents a higher chemosensitivity in early morning and late afternoon when its feeding activity is the greatest (Van der Goes van [START_REF] Van Der | Olfactory sensitivity in tsetse flies: a daily rhythm[END_REF]. The recent application of underwater electroantennography [START_REF] Machon | New electroantennography method on a marine shrimp in water[END_REF]) offers new perspectives of chemosensitivity surveys of our model crustaceans that face various chemical cues at different period of the day. Moreover, the behavioural responses might also be correlated to the expression level of some chemo-receptor such as IR25a [START_REF] Zbinden | Comparative Study of Chemosensory Organs of Shrimp From Hydrothermal Vent and Coastal Environments[END_REF] in order for the crustacean antennules to perceive chemical cues. For technical reason, most of our morning behavioural experiments could only be conducted during daylight hours and we believe that the combination of additional experiments performed in the darkness combined to chemosensitivity surveys might further support our hypothesis on the role of the chemical seascape on diurnal migrations of cave-dwelling crustaceans.

Very few studies in chemistry have attempted to identify the structure of the hypothesized marine chemical cues involved in habitat selection [START_REF] Lecchini | Larval sensory abilities and mechanisms of habitat selection of a coral reef fish during settlement[END_REF][START_REF] Derby | Neural Processing, Perception, and Behavioral Responses to Natural Chemical Stimuli by Fish and Crustaceans[END_REF][START_REF] Lecchini | Ecological determinants and sensory mechanisms in habitat selection of crustacean postlarvae[END_REF][START_REF] Lecchini | Transmission distance of chemical cues from coral habitats: implications for marine larval settlement in context of reef degradation[END_REF]. Even though we expected to detect some of the previously known compounds of the four sponge models in the SCS, our metabolomics approach shows that none of the specialized metabolites reported so far from the global metabolome of our sponge models could be found in the SCS. Based on their molecular formulae as obtained unambiguously by HRMS, the chemomarkers present in the sponge conditioned seawater, and at low concentration in the cave seawater, correspond to C18-based oxylipins. Further confirmation of these structures is needed, however, using available standards for example. This hypothesis seems realistic as diatom oxylipins are known for their multiple functions, such as sexual pheromones or food-finding cues detectable by grazers [START_REF] Fink | Oxylipins from freshwater diatoms act as attractants for a benthic herbivore[END_REF][START_REF] Kâ | Impact of the diatomderived polyunsaturated aldehyde 2-trans,4-trans decadienal on the feeding, survivorship and reproductive success of the calanoid copepod Temora stylifera[END_REF], but also as chemical-alarm cues when facing grazing pressure [START_REF] Ianora | Aldehyde suppression of copepod recruitment in blooms of a ubiquitous planktonic diatom[END_REF][START_REF] Pohnert | Diatom/copepod interactions in plankton: the indirect chemical defense of unicellular algae[END_REF]. Thus, the ecological function of oxylipins may be very broad. The putative oxylipins may be produced by any of the sponge holobionts [START_REF] Mcclintock | Ecology of Antarctic marine sponges: an overview[END_REF]) and they may be responsible for the homing behaviour of crustaceans. The four peptides naturally found in high concentrations in the cave seawater are also likely produced by sponge holobionts, although in this case their very low concentrations in the sponge conditioned seawater do not provide information on their putative function in the cave chemical seascape.

In addition to the organic compounds in the seawater which are detectable by our metabolomics approach, we assume that inorganic compounds may also influence the biological responses of our model organisms. A significant difference in nutrient concentrations between seawater treatments would only result from the fluxes generated by the sponge holobiont catabolism (for a review see [START_REF] Maldonado | Nutrient fluxes through sponges: biology, budgets, and ecological implications[END_REF], releasing ammonia, nitrate or phosphate for instance during the water conditioning. However, as the sponge volume used in our experiments represented only 1% of the aquarium volume, we can reasonably consider that there was a very low probability of a significant change in nutrient concentration during the preparation of our seawater treatments.

Finally, elucidating the mechanisms and strategies used in habitat detection is crucial to predict how individual species or assemblages may respond to the degradation of their environment [START_REF] Swihart | Responses of 'resistant' vertebrates to habitat loss and fragmentation: the importance of niche breadth and range boundaries[END_REF][START_REF] Jones | Coral decline threatens biodiversity in marine reserves[END_REF]). Changes affecting chemical mediation processes are likely to impact subsequent distribution of marine organisms. Sponges are known to be sensitive to global changes, and most of our study species have already suffered mass mortalities related to extreme climate events [START_REF] Lejeusne | Climate change effects on a miniature ocean: the highly diverse, highly impacted Mediterranean Sea[END_REF]. In a context of increasing frequency of such events [START_REF] Giorgi | Climate change projections for the Mediterranean region[END_REF], we can expect further alteration of sponge communities to impact distance interactions mediated by sponge chemical cues. Underwater caves are fragmented habitats and their biodiversity patterns are greatly shaped by their connectivity. Therefore, any alteration of the chemical seascape within and surrounding marine caves, directly or indirectly caused by human activities, could have severe consequences on these ecosystems. As has been evidenced in previous studies by [START_REF] Dixson | Coral reef fish smell leaves to find island homes[END_REF][START_REF] Dixson | Terrestrial chemical cues help coral reef fish larvae locate settlement habitat surrounding islands[END_REF], connectivity between distinct marine habitats may partly rely on chemical mediations, and not only on currents.

Figure legends

Fig. 1

 1 Fig. 1 Response of the four crustaceans to: (a) underwater cave seawater (+CS) vs. control

Fig. 2

 2 Fig. 2 Response of Hemimysis margalefi and Leptomysis sp. to the underwater cave seawater

Fig. 3 Fig. 4

 34 Fig.3Response of the four crustaceans to sponge conditioned seawater (SCS) vs. control

Fig. 5

 5 Fig. 5 Principal Component Analysis (PCA) based on the chemical features of the different

Fig. 6

 6 Fig. 6 Heatmap based on the variable importance in projection of the features (VIP > 1) detected

Fig. 7

 7 Fig. 7 Comparative ion intensities of the features corresponding to the chemical cues A-D (1st

ACKNOWLEDGEMENTS

The authors are grateful to the TOTAL Foundation for funding our research programme.

This work was performed in the framework of the French-Brazilian International Associated Laboratory "LIA MARRIO" of the CNRS, and received the support of the MALLABAR metabolomics plateau funded by the CNRS, the PACA Region and ANR. We warmly thank Pierre Chevaldonné, Cesar Ruiz and Laurent Vanbostal for collecting H. margalefi and Leptomysis sp. individuals. We are also grateful to Philippe Aublanc of the Paul Ricard Oceanography Institute for providing P. serratus and P. elegans individuals, to Eva Ternon for her assistance to the seawater processing prior to metabolomics, to Jean Charles Martin for his contribution to the development of bioinformatics tools, and to Rachel Mackie for her reviewing of the English.

AUTHOR CONTRIBUTION STATEMENT

MS and TP designed the experiments. MS and MLC performed the experiments. MS, SG, MLC and OPT analysed the data. MS, SG, MLC, OPT and TP wrote the manuscript.

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.