In Situ Capture and Real-Time Enrichment of Marine Chemical Diversity

Abstract

Analyzing the chemical composition of seawater to understand its influence on ecosystem functions is a long-lasting challenge due to the inherent complexity and dynamic nature of marine environments. Describing the intricate chemistry of seawater requires optimal in situ sampling. Here is presented a novel underwater hand-held solid-phase extraction device, I-SMEL (In Situ Marine moleculELogger), which aims to concentrate diluted molecules from large volumes of seawater in a delimited zone targeting keystone benthic species. Marine benthic holobionts, such as sponges, can impact the chemical composition of their surroundings possibly through the production and release of their specialized metabolites, hence termed exometabolites (EMs). I-SMEL was deployed in a sponge-dominated Mediterranean ecosystem at a 15 m depth. Untargeted MS-based metabolomics was performed on enriched EM extracts and showed (1) the chemical diversity of enriched seawater metabolites and (2) reproducible recovery and enrichment of specialized sponge EMs such as aerothionin, demethylfurospongin-4, and longamide B methyl ester. These EMs constitute the chemical identity of each targeted species: Aplysina cavernicola, Spongia officinalis, and Agelas oroides, respectively. I-SMEL concentrated sponge EMs from 10 L of water in a 10 min sampling time. The present proof of concept with I-SMEL opens new research perspectives in marine chemical ecology and sets the stage for further sustainable efforts in natural product chemistry.

Figure 1

Representation of the I-SMEL instrument and its deployment in a Mediterranean marine ecosystem. Schematic view of the main functioning items (a) and views of the disassembled (b) and assembled (c) instrument (1. electronic controller of the pump, 2. peristaltic pump, 3. valves, 4. SPE ports, 5. pump outlet to connect a flexible tank, and 6. battery). See also S1 for more details. I-SMEL operated by a SCUBA diver in a coralligenous community (d) and then above the studied sponges (e, f). In situ pictures of the three targeted sponge species, Aplysina cavernicola (g), Agelas oroides (h), and Spongia officinalis (i).

Figure 2

Sample preparation and analytical pipeline to characterize the structural diversity of captured marine exometabolites (EMs) and determine the proportion of sponge-specialized EMs. SO: Spongia officinalis. AC: Aplysina cavernicola. AO: Agelas oroides. HR-MS²: High-resolution tandem mass spectrometry. SPE: Solid-phase extraction.

Figure 3

Chemical diversity of captured EMs. (a) Feature-based molecular network with ion identity containing 805 nodes organized in 137 spectral families (see also S4). The size of the node is proportional to the retention time. The pie chart represents relative intensities of features (FT) in each sample (ACS = average chemical seascape, AO = Agelas oroides, SO = Spongia officinalis, and AC = Aplysina cavernicola). (b) FT distribution and chemical diversity based on natural product pathway probabilities (>0.8) determined with CANOPUS. Sponge EM extracts contained three types of FT: identical to those from their crude extract (type 1), uniquely found as sponge-specific EMs (type 2), and clustering with features from ACS (type 3 in green). (c) Total number of FT for each sponge species and their distribution showing reproducibly captured EMs in sampling replicates (EXP2-3).

Figure 4

Spectral families and structural diversity of reproducibly enriched A. cavernicola specialized EMs. (a) Focus on the major bromo-spiroisoxazoline spectral family with the reproducibly detected features used for structural dereplication. Each feature is identified by its m/z (M) and retention time (T). The pie chart represents relative intensities of features in each sample. (b) Focus on four other spectral families, each containing one brominated alkaloid detected in both crude and EM extracts (confidence of identification level 2b for molecules associated with a star). (c and d) The relative concentration of each annotated EM was determined by measuring the area under the curve (AUC) of extracted ion chromatograms (EIC) in each extract and using the crude extract as reference (100%). (d) Metabolites more concentrated in EM extracts than in the crude extracts.

Figure 5

Spectral families of reproducibly released Spongia officinalis EMs. Each feature is identified by its m/z (M) and retention time (T). The pie chart represents relative intensities of features in each sample. (a) Focus on the furanoterpenoid spectral families with MS² spectra of putative furospongin-1 and demethylfurospongin-4 showing a characteristic fragment at m/z 135.0800 corresponding to the furan fragment [C₉H₁₀O + H]⁺. Structural dereplication was performed in agreement with previously reported data^, (confidence level 2b). (b) M353T21.1 is an in-source fragment of the same molecule as M430T21.1, a scalarane sesterterpenoid. (c) The relative concentration of each annotated EM was determined by measuring the area under the curve (AUC) of extracted ion chromatograms (EIC) in each extract (EXP2) and using the crude extract as a reference (100%).

Figure 6

Spectral families representing the reproducibly enriched specialized EM from Agelas oroides. Each feature is identified by its m/z (M) and retention time (T). The pie chart represents the relative intensities of features in each sample. (a) Main spectral family related to AO and containing the oroidin MS² spectrum. (b) Putative longamide B methyl ester, clustering with longamide B, is reproducibly detected as EM around the sponge (confidence level 2b). (c) The relative concentration of such EM is twice lower on average than in the crude extracts.