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Chemical mediation is the most widely used mode of communication in the living world and is commonly considered as the “language of life” (Wyatt, 2010, 2014). Exchanges of molecules as chemical cues between organisms are involved in reproduction, defense or feeding and therefore, significantly contribute to the organization and functioning of ecosystems (Burks and Lodge, 2002; Hay, 2009; Hentley and Wade, 2016). Pollination is one of the most famous examples of a chemosensory-mediated network of biotic interactions, plant odors thus structuring the biodiversity of a plethora of arthropods and flying vertebrates (Parachnowitsch and Manson, 2015). More generally, chemical mediation structures the spatial distribution of many animal species capable of orienting themselves within a complex mosaic of odors, often defined as a chemical landscape (Dyer et al., 2018). In the marine environment, the potential existence of a similarly complex chemical seascape, made of a great diversity of molecules transported by water currents, is highly probable. Many of these molecules originate from the metabolic activities of marine organisms. Exudated in the surrounding water, they are named exometabolites and they may carry potential signals for the whole ecosystem (Cassier et al., 2000; Mauduit et al., 2023).

Several studies highlighted that the recruitment of marine species requires the use of chemical cues. For instance, Atema et al. (2002) presented the first evidence that larval reef fishes (primarily apogonids) were able to distinguish between the chemical seascapes from the lagoon and the open ocean. Lecchini et al. (2013) also observed that several fish, crustacean and cephalopod larvae actively selected settlement habitats, assuming the use of chemical cues. The presence of conspecifics, food, the nature of the biodiversity assemblage or the healthy state of an ecosystem, all produce cues susceptible to attract juveniles to a suitable environment for settlement. For example, Coppock et al. (2013) found that juvenile reef fishes exhibited a significant preference for the water conditioned by conspecifics or by some reef-building species. Similarly, Lecchini et al. (2014) showed that various marine larvae were significantly more attracted by the chemical seascape of living corals than by that of dead colonies.

Depending on their fate (e.g. adsorption on particles, solubility), molecules may be transported over long distances and be perceived by chemo-sensitive organisms (Mollo et al., 2014, 2017). Lecchini et al. (2014) found that molecules emitted by corals can be transported over 2 km in the ocean and that they could be detected more than 1 km away by fish larvae searching for their recruitment habitat. Distant chemical cues also mediate habitat selection for migratory species such as salmonid fish and turtles (Stabell, 1992; Endres et al., 2016). They follow chemical corridors to accurately return to their natal habitat, a specific behavior known as homing (Scholz et al., 1976).

However, none of these pioneer studies in marine chemical ecology has addressed the chemical nature of these seascapes, influencing the behavior of many mobile marine organisms. Access to this chemical information is even more crucial as global change alters the environmental quality, and thus, putatively chemical mediation and ecosystem functioning (Munday et al., 2009; Saha et al., 2019; Wilke, 2021; Roggatz et al., 2022). Advances in mass spectrometry (MS)-based metabolomics, including data processing and spectral annotation, have improved the characterization of mixtures of unknown molecules originating from different environments, such as seawater. The knowledge gained from such analyses greatly benefits marine chemical ecology research and provides new opportunities to understand the marine ecosystem functioning (Santonja et al., 2018; Wegley Kelly et al., 2022; Mauduit et al., 2023; Nelson et al., 2023; Quinlan et al., 2023).

Underwater cave ecosystems allow working on simplified processes compared to the open sea, thus providing experimental conditions such as natural mesocosms to study chemical mediation. Of reduced size, they provide a stable environment with little or no light or water movement leading to a low food supply (Harmelin et al., 1985). Among the most successful inhabitants of marine caves, sponges (Porifera) are known to produce a great diversity of metabolites (Carroll et al., 2022), some of them being released into the environment (Ternon et al., 2016; Mauduit et al., 2022, Mauduit et al., 2023) and contributing to a complex chemical seascape. Mobile dwellers of the cave ecosystem include teleost fishes and various crustaceans, such as mysids (Crustacea: Mysida) (Rastorgueff et al., 2011). Mysids can form dense swarms with millions of individuals in a single cave (Passelaigue and Bourdillon, 1986; Coma et al., 1997; David et al., 1999).

Cave mysids including Hemimysis species display circadian horizontal migrations outside their caves to find food and avoid predation at night (Macquart-Moulin and Passelaigue, 1982; Passelaigue and Bourdillon, 1986; Riera et al., 1991). At the end of the night, they return from the open-sea to their cave habitat. In their study, Santonja et al. (2018) used the context of underwater marine caves to address the potential role of sponge-produced metabolites on the migratory behavior of Hemimysis margalefi. MS-based metabolomics led to the determination of molecular formulae of chemical cues suspected to participate in the swimming behavior of cave mysids as well as of shrimps (Crustacea: Decapoda). These compounds were found both in experimental waters potentially containing sponge exometabolites and also naturally occurring in seawater collected in an underwater cave. They were thus hypothesized as possible chemical cues guiding cave mysids during their nocturnal migrations. Furthermore, as the behavioral responses were most pronounced during morning experiments, Santonja et al. (2018) hypothesized that sponge metabolites might act as recognition cues when H. margalefi return to their cave at dawn. Given that each marine cave harbors an original combination of sponge assemblages (Grenier et al., 2018) and that H. margalefi populations display high spatial genetic structuring (Lejeusne and Chevaldonné, 2006; Rastorgueff et al., 2014), it could even be conceivable that a given H. margalefi population could recognize the chemical composition of its own cave water. This would constitute an interesting homing behavior (Brönmark and Hansson, 2000), a case worth of investigation.

The aim of the present study was to evaluate individual cave seawater recognition by cave-dwelling mysids and to compare the overall chemical composition of each tested cave seawater in an attempt to correlate a choice behavior with reproducibly detected molecules. The behavioral response of different populations of two mysid species was evaluated on seawaters naturally occurring in three different underwater caves. The selected species are the cave-dwelling H. margalefi and a non-cave mysid, Leptomysis sp. living in nearby shallow water environments. Hence, the two species have contrasting ecological habits.

Previous studies highlighted the chemical influence of conspecifics on habitat selection for crustaceans such as barnacle larvae (Matsumura et al., 1998), shrimp larvae (Lecchini et al., 2017) and spiny lobsters, where sex pheromones such as conspecific urine of both sexes acted as aggregation signals (Horner et al., 2006). Considering the gregarious behavior of mysids, one might conceive that conspecific chemical signals could play a role in orienting H. margalefi migrations. Therefore, we also tested their choice behavior on seawater originating from aquaria where the same species of mysids were kept. To conduct the experiments, we combined the use of (i) an experimental two-choice device to assess the behavioral response of the crustaceans, and (ii) MS-based metabolomics to decipher the chemical composition of each tested seawater condition.

Previous studies on Mediterranean caves led to the proposal of a first functioning model based on trophic exchanges (Russo and Bianchi, 2003; Rastorgueff et al., 2011, 2015). In this model, crustaceans living in dense swarms, such as mysids, are a keystone component driving organic matter transfers from the open sea to the back of the caves, thus mitigating the usual oligotrophy of the ecosystem. These horizontal migrations are similar to the vertical migrations of zooplankton (Hutchinson, 1967; Hays, 2003) and some pelagic mysid species (Viherluoto and Viitasalo, 2001) occurring at night. They navigate between the upper water column and deeper zones, a behavioral strategy to limit predation pressure while accessing to food resources (Lampert, 1993). Through their circadian migrations, whether vertical or horizontal, these small crustaceans are also vectors of organic matter supplying lower and upper levels of the trophic network (Rastorgueff et al., 2011; Kelly et al., 2019), thus playing an important role in the ecosystem functioning.

Few studies investigated the mechanisms behind the swimming behavior of cave-dwelling mysids. Previous laboratory observations (Macquart-Moulin, 1979; Bourdillon et al., 1980) showed that the movements of H. speluncola (a species closely related to H. margalefi, also found at 3PP cave) responded to light intensity. After in-situ experiments, Passelaigue and Bourdillon (1986) and then Riera et al. (1991) confirmed that light was the initial factor triggering the migration of H. speluncola. Other environmental factors could also influence the orientation (geotaxis) of migratory mysids such as temperature variation (Bourdillon and Castelbon, 1983) and current velocity (Crouau, 1986). Some studies also reported that chemical cues could influence mysid swimming behavior (Mauchline, 1980; Hamrén and Hansson, 1999). As chemical cues are among the main detection mechanisms involved in marine habitat recognition, notably in crustaceans (Boudreau et al., 1993; Castañeda-Fernández De Lara et al., 2005; Arvedlund and Kavanagh, 2009; Lecchini et al., 2010), it could be assumed that chemical mediation could also be involved in the habitat recognition of H. margalefi.

Santonja et al. (2018) tested this hypothesis in a previous study and showed that H. margalefi could significantly recognize a cave seawater or a seawater conditioned by syntopic cave-dwelling sponge species compared to a control seawater. This study suggested the possible involvement of recognized chemical signals from the mysid’s habitat. The present study intended to further investigate whether this recognition could be considered as a homing behavior and whether each cave displayed a singular chemical seascape that could play a role.

These new series of experiments showed that a given mysid population could recognize its home cave seawater. Furthermore, they showed no preference when exposed to a seawater from a different cave and a control seawater. These results suggest that the circadian migrations of H. margalefi, although most likely triggered by light, could use chemical recognition to ensure that mysids return to their home cave. Such homing behavior consequently constrains genetic exchanges with individuals from other caves. The consequences of homing behavior on the genetic structure of populations have already been demonstrated for salmons (Neville et al., 2006) and reef fishes (Gerlach et al., 2007; Salles et al., 2016) and could also contribute, in addition to parental brooding and natural habitat fragmentation, to explain the strong structuring of H. margalefi populations across Mediterranean caves (Lejeusne and Chevaldonné, 2006; Rastorgueff et al., 2014). To further explore the interactions between habitat recognition and genetic structuring of populations, it would be interesting to compare the behavioral responses of mysid populations with different degrees of genetic similarity (e.g. belonging to different caves within a single metapopulation or across genetic boundaries).

In the present study, we did not observe differences in behavioral responses between morning and afternoon, thereby suggesting that although H. margalefi homing must occur before daybreak, habitat chemical recognition might be independent of the time of the day. This result contrasts with those of Santonja et al. (2018) who recorded a greater capacity for habitat recognition during the morning experiments. Reproducibility of behavioral responses through time is a recurring problem in chemosensory experiments (Stamps et al., 2012; O’Neill et al., 2018; Hardege et al., 2024). Variations in time are anticipated among wild populations over the years separating our experiments. For example, microevolution in wild salmons can occur rapidly in response to global warming (Kovach, 2012; Piou and Prevost, 2013). In this sense, Kovach et al. (2012) demonstrated significant changes in genetic diversity associated to late-migration timing after only 17 generations. For H. margalefi, 4 cohorts are produced per year (Lejeusne and Chevaldonné, 2005), resulting in frequent turnover of the local population, which is increasingly exposed to environmental changes. This turnover may lead to small changes in the behavior of mysids, although the main migratory pattern is conserved over time.

Conspecific exometabolites in the seawater do not seem to influence the behavior of H. margalefi during its migrations. This result is in line with those from Lecchini et al. (2010), who showed that out of 9 studied crustaceans, 6 performed an active habitat selection for settlement without considering the presence or absence of conspecifics. They also found that in the case of conspecific attraction, visual cues are rather mobilized. These results strengthen the hypothesis of a homing behavior that could result from the detection of specific signals in the chemical seascape of their habitat.

Each of the three caves studied here harbors a specific chemical seascape. 3PP seawater is the most diverse, but also the one with the largest number of organic pollutants such as the alkyl ethoxy sulfate compound ( Supplementary S6.3 ). This compound is a surfactant notably used as detergent and has been detected in river sediments after being released into the environment via wastewater (Sanderson et al., 2006). Its putative presence in the 3PP seawater samples could be explained by the proximity of the sewage outlet of La Ciotat city. Conversely, no organic pollutant was reproducibly found in Jarre, potentially because this cave, located on an island, has its entrance towards the offshore and is therefore less exposed to pollution. A few reproducibly detected compounds could derive from biological origin belonging to four chemical class: peptide, fatty acids, steroids and alkaloids. In crustaceans, peptides are reported to have signaling functions to coordinate homing and settlement (Rittschof and Cohen, 2004). Here, the peptide annotated as cyclo(leucylleucyl) has been reported in several bacterial taxa, including microbes associated with macroalgae (Anjali Das et al., 2023) but no ecological role has been associated with it so far. Lipids (Sakata et al., 1988) and steroids (Dulka et al., 1987) could also act as marine chemical cues. The sulfated fatty acid (molecular formula: C₃₂H₆₅NO₆S) detected in Jarre may be a sphingolipid known from Cytophaga johnsona (Abbanat et al., 1988), bacteria commonly found in marine environments (Reichardt et al., 1983). In 3PP seawater, the reproducibly found steroid identified as a sulfated cholesterol (molecular formula C₂₇H₄₆O₄S), represents a family of compound known from various benthic macroorganisms such as sea cucumbers, brittle stars (Levina et al., 1991; Makarieva et al., 1993) and sponges (Dai et al., 2010; Mioso et al., 2017). For instance, the amaroxocane B is a sulfated sterol produced by the coral reef sponge Phorbas amaranthus and interestingly, the Mediterranean sponge Phorbas tenacior is a common inhabitant of cave entrances (Grenier et al., 2018).

Santonja et al. (2018) hypothesized that 4 peptides and 4 oxylipin derivatives, potentially released by sponge holobionts, could play a major role in the habitat recognition for H. margalefi. In our dataset, we found 5 of these compounds, but they were neither reproducibly found (i.e. not present in all cave seawater replicates) nor specific to a particular cave ( Supplementary S7 ), and thus did not appear in Figure 6 . For instance, the peptide C₂₄H₄₄N₄O₄ was detected in Jarre (1/6 samples) and Fauconnière (1/6 samples) but never in 3PP seawater. Two oxylipins, C₁₈H₃₄O₅ and C₁₄H₂₆O_3, were found in 3PP (1/4 samples) and Fauconnière (1/6 samples and 3/6 samples) but never in Jarre seawater. Moreover, 2 compounds were detected in more than a half of the replicates of each of the 3 cave seawaters. The putative peptide C₄₂H₇₇N₇O₇ was reproducibly found in all seawater replicates of the 3 caves. The oxylipin C₁₈H₃₀O₃ has been detected in 3PP (1/4 samples), Fauconnière (1/6 samples) and in Jarre (4/6 samples) but below the detection intensity limit (1E², whereas the noise level for MS¹ detection was fixed at 5E³). These last two compounds could thus be considered as cave chemical markers. The difficulty to reproducibly find the same dissolved compound, from one sample to another, has already been reported by Mauduit et al. (2023). In their study, they suggest that possible biotransformations could take place in the seawater for some structural families. Indeed, oxylipins are polyunsaturated fatty acids, which once dissolved in the seawater, may be subject to oxidation or hydroxylation of their carbon chain.

In conclusion, the present study provides new evidence that the migratory behavior of H. margalefi might mostly refer to daily-based homing rather than to an occasional habitat recognition. We suggest that this behavior could be related to a specific chemical recognition which is not due to the involvement of H. margalefi’s exudates, but rather to the presence of a singular combination of compounds from its home habitat. Each unique chemical seascape is composed of a complex mixture of compounds belonging to different structural classes, of possible natural origin. A more precise characterization of these chemical seascapes is difficult to achieve because a majority of compounds are present in trace amounts and the fate of the majority of biosynthesized metabolites in seawater is yet unknown. The referencing of marine structural diversity is widely documented in various molecular databases (e.g. Marinlit, Lotus). Accurate identification of already known molecule requires access to raw spectral data both MS and NMR. The paucity of freely available spectral data of marine natural products slow down the dereplication process (syn. Confirming the identify of already known compounds). Such identification is further complicated by seawater biotransformation that a waterborne metabolite may undergo. Freely available marine spectral databases are urgently needed to help decipher further the exometabolite composition of seawater.

Determining the origin of biosynthesized molecules and their contribution to chemical seascapes is truly crucial. The availability of new devices enabling to concentrate diluted molecules and to trace fingerprints of keystone species represent a new hope to show how chemical mediation plays a central role in marine ecosystems (Mauduit et al., 2023). In the current context of global change and major threats on marine biodiversity, any change in species composition and/or abundance (e.g. sponges or corals frequently subject to mortality events) is likely to impact the composition of the chemical seascape and thus marine ecosystem functioning.

The Supplementary Material contains further details on the experimental details for MS-based Metabolomic analyses, responses of mysids according to the time of the day (morning vs. afternoon) with detailed statistical results, Partial Least Squares Discriminant Analysis (PLS-DA) comparing chemical features detected in the control and cave seawater samples, detailed statistical results of PLSDA analysis of Figure 5A , the merged feature-based molecular network combining data acquired in both positive and negative modes with publicly accessible job ID, MS data of reproducible features with proposed structural classes and MS data of the compounds found in seawater from both our previous (Santonja et al., 2018) and current studies.