Experimental evidence of mixotrophy in seagrass-associated Lucinidae in the absence of sulfide

Experimental evidence of mixotrophy in seagrass-associated Lucinidae in the absence of sulfide | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Experimental evidence of mixotrophy in seagrass-associated Lucinidae in the absence of sulfide Samuel Orgeas-Gobin, Alexandre Nguyen-tiet, Lucie Blondel, Benjamin Marie, and 3 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-9360400/v1 This work is licensed under a CC BY 4.0 License Status: Under Review Version 1 posted 4 You are reading this latest preprint version Abstract Bivalves of the family Lucinidae, Loripes orbiculatus and Lucinoma borealis , are sympatric species inhabiting coastal seagrass beds in Roscoff Bay. These bivalves harbor chemoautotrophic symbionts within their gills that provide autotrophic nutrition to the host by oxidizing hydrogen sulfide (H₂S) present in the sediment. Although Lucinidae are typically considered fully autotrophic in these environments, seagrass beds are subject to fluctuations in sulfide availability due to tides, seasonal changes, and anthropogenic disturbances. This study investigates how Lucinidae cope with periods of low sulfide availability by exploring their nutritional strategies under sulfide starvation. Lucinidae species were incubated for 15 days in the presence of sediment bacteria or a mixture of two phytoplankton species labeled with ¹⁵N and ¹³C, with or without addition of sulfide, to trace assimilation pathways into the gill and visceral mass. Results show that both ¹⁵N and ¹³C were incorporated into tissues within seven days, indicating that lucinids are capable of assimilating both autotrophy- and heterotrophy-derived sources of nutrition. Composition of their associated bacterial communities was not affected. These findings provide evidence of mixotrophy in coastal Lucinidae, indicating that they can shift to filter-feeding under low sulfide availability, probably contributing to their ecological success. Nutritional plasticity of the Lucinidae may be key to their resilience in fluctuating coastal environments. Isotopy Mixotrophy Seagrass bed Lucinoma borealis Loripes orbiculatus Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Introduction Lucinidae bivalves harbor chemosynthetic bacterial symbionts and inhabit seagrass beds and mangroves habitats, among others, where oxic-anoxic interfaces occur near the rhizosphere (Dubilier et al, 2008 , Osvatic et al., 2023 ). Seagrass beds are characterized by high organic matter inputs from plant litter, and strong biogeochemical fluctuations. In the underlying oxygen-depleted sediments, this organic matter is degraded by sulfate-reducing bacteria, which use sulfate as an electron acceptor and produce H₂S as a by-product (Jørgensen, 1977; Boudreau & Westrich, 1984). Reduced sulfur compounds, notably H₂S, serves as a substrate for the chemosynthetic Gammaproteobacteria occurring in the gill bacteriocytes of Lucinidae (Dando et al., 1986; Johnson & Fernández, 2001; Pales Espinosa et al., 2013 ). Plant roots also release oxygen into the surrounding sediment (Sand-Jensen et al., 2005; Heide et al., 2012; Sanmartí et al., 2018), supporting the activity of lucinids and symbionts and promoting plant growth and ecosystem stability. Besides sulfur oxidation, genomes of some symbionts also encode nitrogenase, enabling biological nitrogen fixation, which may benefit both the host and the plant by alleviating the nitrogen limitation inherent to seagrass beds (Petersen et al., 2017; König et al., 2017; Cardini et al., 2019). Because sulfide is toxic to both animals and plants, Lucinidae symbioses also contribute to detoxification of the host environment and rhizosphere. For this reason, authors have proposed the hypothesis of a tripartite symbiosis between seagrass-bed, Lucinidae and Lucinidae-associated symbionts (Distel, 1998; Van der Geest, 2013). Seagrass beds-associated Lucinidae are considered as primarily relying on symbiont-derived organic compounds. However, environmental fluctuations (e.g., tides, sunlight, temperature) and anthropogenic pressures (e.g., global changes, eutrophication, sediment resuspension) cause variations in H₂S availability, resulting in alternating low- and high-sulfide periods (Kock et al., 2022). During winter, decreased temperature and light reduce both microbial and photosynthetic activity, lowering sulfide production. This raises questions about the persistence of chemosynthetic nutrition and the fate of symbionts (Brodersen et al., 2017 ; Shrameyer et al., 2018). Conversely, in nutrient-rich agricultural regions, fertilizer runoffs promote algal and microbial blooms, increasing organic matter degradation and H₂S production (Neufeld et al., 2002 ; Thorsen et al., 2019 ). These shifts can occur rapidly, requiring some level of plasticity to deal with changes. Several studies have shown that mangrove- and seagrass-associated Lucinidae can survive extended periods of sulfide starvation (Caro et al., 2009 ; Elisabeth et al., 2014 ; Orgeas-Gobin et al., 2025 ), displaying symbiont density decline. To compensate, Lucinidae may supplement their nutrition through heterotrophy. For example, gut content analyses of Lucinoma borealis revealed ingested diatoms, suggesting facultative heterotrophy (Dando et al., 1986). In Loripes orbiculatus , seasonal changes in the ratio of bacteriocytes to mucocytes in gill tissues also suggest potential shifts in nutritional mode (Roques et al., 2020). In this study, we test the hypothesis that coastal lucinids are mixotrophic, supplementing autotrophy with filter-feeding during low sulfide periods. For this, specimens of Loripes orbiculatus and Lucinoma borealis were collected during winter to assess their natural C and N isotopic signatures during a presumed low-sulfide period. To test their potential for filter-feeding, specimens were then experimentally exposed to 13 C- and 15 N-labeled cultures of either bacteria or larger phytoplankton, representing different types of potential prey, with or without a sulfide source. Potential shifts in symbiont community composition were investigated by analyzing bacterial community composition using 16S rRNA metabarcoding, and changes in holobionts functional profile were explored using non-targeted metabolome analysis. Results from both lucinid species, as well as the organization of symbionts Rubisco-encoding genes, were compared. To our knowledge, this study provides the first experimental evidence of a filter-feeding strategy in lucinids. Material and methods Lucinidae Collection Two groups of Lucinidae were collected at different periods for two distinct analyses: the natural isotopic signature analysis (2020 Lucinidae group) and the feeding experiment (2023 Lucinidae group). Specimens were collected in the sublittoral area adjacent to a Zostera marina seagrass bed near the Roscoff Biological Station (Roscoff, France; 48.7314 N, -4.0024 W; sediment depth: 20 cm). For the natural isotopic signature measurement, a total of 30 Lo. orbiculatus , 10 Lu. borealis and 3 Tellina sp. were collected. Tellina sp. is a non-symbiotic heterotrophic bivalve that lives in sympatry in seagrass-beds (heterotrophic control). Upon collection, clams were dissected under sterile conditions as follows: hemibranchs and visceral mass were flash-frozen separately in liquid nitrogen and stored at -80°C until mass spectrometry (MS) analysis. For the feeding experiment, 120 Lo. orbiculatus and 120 Lu. borealis were collected in February 2023. Directly upon collection, seven individuals per species were dissected under sterile conditions (T₀ environmental samples, natural isotopic reference samples) and compared with those collected in February 2020. The remaining individuals were maintained in aquaria at the Roscoff Marine Biological Research Center (CRBM). During feeding experiments, all Lucinidae were dissected as follows: the two hemibranchs and the remaining visceral mass were flash-frozen separately in liquid nitrogen and stored at − 80°C. One hemibranch and the visceral mass of each individual were used to assess isotopic composition via mass spectrometry, while the second hemibranch was used for sequential metabolite and DNA extraction for metabolomic and metabarcoding analysis. Microorganism cultivation and stable isotopic labeling For the feeding experiment, two cultures of microorganisms were produced: one consists of environmental bacteria isolated from seagrass bed sediments, and the second is a mixture of two phytoplankton strains, Tisochrysis lutea RCC¹³⁴⁹ and Chaetoceros calcitrans RCC¹⁸⁰⁹, obtained from the Roscoff Culture Collection (RCC). Bacteria were directly sampled from seagrass bed sediments at a depth of 20 cm. The sediments were inoculated into Marine Broth 2216 medium (Millipore, Burlington, MA, USA), and incubated for seven days at 14°C in the dark until reaching the stationary phase. Phytoplankton strains were cultured separately under sterile conditions (24 h light, 20°C) in 10 L oxygenated flasks. T. lutea was grown in sterile seawater supplemented with 1 mL/L of Conway medium (Lananan et al., 2013) and bubbled with CO₂ until reaching the stationary phase (seven days). C. calcitrans was cultivated in a mixture of sterile seawater (2/3) and freshwater (1/3), also supplemented with 1 mL/L of Conway medium and bubbled with CO₂, reaching the stationary phase after five days. Both phytoplankton strains were then mixed together. After microorganism cultivation, two distinct feeding mixture were obtained, the environmental bacteria and the phytoplankton cultures. Once both microorganism cultures reached a density of 10⁶ CFU/mL, 5 L of each were harvested and stored at 4°C to halt further growth. A subsample of each unlabelled culture was used to determine the baseline isotopic signatures (control samples). The remaining cultures were then labelled by adding 500 mL of a 4 mM ¹³C solution (NaH¹³CO₃; 99% ¹³C) and 500 mL of a 1 g·L⁻¹ ¹⁵N solution (¹⁵NH₄Cl; 98% ¹⁵N; Eurisotop, Saarbrücken, Germany), and incubated for four days. These concentrations were selected based on previous isotopic labelling experiments on microalgae (Leroy et al., 2012 ). Following labelling, cultures were centrifuged twice (15 minutes, 7000 g, 20°C) to remove residual non-incorporated isotopes and terminate further incorporation. The resulting pellets were resuspended in sterile seawater and stored at 4°C until further use. A fraction of each labelled culture was preserved to assess the post-labelling isotopic enrichment. Elemental analysis shows that microorganisms sources have been well enriched: T. lutea δ 13 C = 598.8‰ and δ 15 N = 195,948.7‰, C. calcitrans δ 13 C = 394.1‰ and δ 15 N = 223,535.3‰, environmental bacteria δ 13 C = 14,494.8‰ and δ 15 N = 525.3‰. Feeding experiment Six 3-L aquaria were set up room, each containing 2 L of 0.22 µm-filtered seawater (water temperature = 14°C, salinity = 35 PSU, room temperature = 20°C). Aquarium conditions are summarized in Fig. 1 . For experimental conditions, twenty Lo. orbiculatus and twenty Lu. borealis individuals were present in each aquarium. In aquaria supplemented with labelled microorganisms, 300 mL of labelled culture (10⁶ CFU/mL) were added. For aquaria receiving sulfide, two dialysis bags, each containing 1.5 g of Na₂S, were placed centrally among the bivalves to ensure gradual sulfide release. Sampling was performed at days 7 and 15 (T7 and T15). At each time point, seven individuals per species were collected for isotopic analysis via mass spectrometry. Half of the aquarium water was replaced daily, and the dialysis bags containing Na 2 S were renewed. Every two days, 200 mL of labelled microorganisms (2 *10 8 CFU) culture was added. To avoid bias in isotopic analysis, individuals were placed in sterile seawater for 48 h post-experimentation to eliminate non-incorporated isotopes prior to dissection. Mass spectrometry analysis Tissues were flash-frozen in liquid nitrogen and stored at -80°C. To prevent isotopic cross-contamination, natural (EC, LC and LS) and enriched (LB, LBS, LP and LPS) samples were handled in separate laboratories using dedicated equipment. Frozen samples were placed into open 2-mL Eppendorf tubes and dried in an oven at 50°C for 48 hours. Dried, tissues were ground directly in the tubes using a sterile spatula. Between 0.80 and 1.20 mg of powdered tissue was weighed into tin capsules, sealed, and stored until mass spectrometry analysis. Carbon and nitrogen isotopic ratios were determined using a CHN elemental analyzer (ThermoFinnigan 1112 Series) coupled to an isotope ratio mass spectrometer (ThermoFinnigan MAT Deltaplus) via a Finnigan Con-Flo III interface. Data are expressed in δ (‰), representing the deviation relative to international reference standards: Vienna Pee Dee Belemnite for δ¹³C and atmospheric N₂ for δ¹⁵N. Laboratory standards were calibrated using certified reference materials: NBS19 for carbon and IAEA-N3 for nitrogen. The standard deviations of repeated measurements of δ¹³C and δ¹⁵N in lab standards were 0.10‰ relative to V-PDB and 0.05‰ relative to atmospheric nitrogen, respectively. Statistical analyses were conducted in R (R version 4.4.1, 2024-06-14 ucrt, Posit, PBC). Since the enriched isotopic data did not meet the assumptions of normality and homogeneity of variances (as assessed by the Shapiro-Wilk and Levene’s tests, respectively), we applied an exact permutation test (Fisher-Pitman), implemented in the coin package. Multiple comparisons were adjusted using the Benjamini-Hochberg method. This test is recommended for small sample sizes and datasets with high dispersion, as observed in our case. Sequential extraction of metabolites and DNA Metabolite and DNA extractions were performed to analyze the metabolome and microbiome of the same individual following the protocol described in Duperron et al, 2023 . Briefly, samples were suspended in 200 µL of cold UHPLC-grade methanol-water (75:25%), homogenized mechanically (GLH850 OMNI, 25,000 rpm, 30 s), and sonicated (Sonics Vibra-Cell VCX 130, 60% amplitude, 30 s) on ice. Supernatants collected post-centrifugation (15,300 g, 4°C, 10 min) were stored in amber vials at -20°C for LC/MS analysis (see below). DNA was then extracted from pellets using the QIAGEN PowerLyzer PowerSoil DNA Kit (Hilde, Germany) following the manufacturer's instructions and using FastPrep 5G disruption (5×30 s, 8 m/s). An extraction blank was included. Metabolites from hemibranchs of five specimens of Lo. orbiculatus and Lu. borealis were analyzed using ultra-high-performance liquid chromatography (UHPLC; ELUTE, Bruker) coupled with high-resolution mass spectrometry (ESI-Qq-TOF Compact, Bruker). Metabolomic analyses were performed with MetaboAnalyst 6.0, ( https://www.metaboanalyst.ca/ ) using standard parameters for metabolomics data (Pang et al.,2024). Statistical analyses were conducted in R (R version 4.4.1, 2024-06-14 ucrt, Posit, PBC) using PERMANOVA and pairwise-test using euclidean distance with FDR correction. Following DNA extraction, a PCR1 was performed to amplify a fragment of bacterial 16S rRNA-encoding genes on all samples using primers 341F (5′-CCTACGGGNGGCWGCAG − 3′) and 806R (5′-GGACTACVSGGGTATCTAAT − 3′) (Parada et al., 2016 ), following standard instructions. The PCR program consisted of an initial 3-min denaturation at 94°C, followed by 35 cycles with a 45-s denaturation step at 94°C, 1-min hybridization at 55°C, and a 1 min 30 s elongation step at 72°C. Amplification was verified by 1% agarose gel electrophoresis. The PCR1 products were sequenced using Illumina MiSeq 250 × 2 bp at the Genotoul platform (Toulouse, France). Amplicon sequence analysis was performed using the QIIME2 pipeline (Bolyen et al., 2019, version 2022.8). Amplicon Sequence Variants (ASVs) were obtained with the DADA2 algorithm; forward and reverse reads were trimmed at 230 and 220 bp, respectively. The expected error rate was set at 2. Reads with a phred score < 20 and chimeras were discarded. ASVs were then affiliated taxonomically using the SILVA 138.2–99 SSU database (Yilmaz et al., 2014 ) and chloroplast- and eukaryote-affiliated reads were discarded. Further analyses were done using R studio and the phyloseq package (McMurdie and Holmes, 2013 ). Raw sequences are available at NCBI Sequence Read Archive (Bioproject: PRJNA1266712, samples accession number SAMN56450224 to SAMN56450372). Phylogeny and genomic organization of symbionts Rubisco encoding genes Symbionts’ Rubisco sequences were extracted from Lo. orbiculatus and Lu. borealis annotated metagenomes (personal data), aligned with 20 relevant amino acids sequences from the literature using ClustalW, and a phylogenetic tree was reconstructed using the maximum likelihood method (JTT model, substitution type: amino acid, rates among site: G; 4. Tree inference options: Nearest-Neighbor-interchange, thread = 8). Nodes robustness were evaluated based on 1000 bootstrap replicates using MEGA version 11 (Tamura et al., 2021 ). The 3D structure of Rubisco was predicted based on amino acid sequence using Alphafold (Jumper et al., 2021 ). To visualize possible conformation changes between the different symbionts Rubisco small and large chains, overlapping of both subunits 3D representation has been performed using ChimeraX (Meng et al., 2023 ). Results Isotopic signatures of Lucinidae during winter Isotopic measurements of bivalves collected in 2020 show that Tellina sp . has δ¹³C isotopic values of -18.7 ± 0.3‰ in the gills and − 18.7 ± 0.04‰ in the visceral mass (Vm), respectively. The two Lucinidae species exhibit more negative values. Lo. orbiculatus , displays δ¹³C values of − 31,4 ± 1‰ for the gills and − 29.1 ± 0.6‰ for the Vm. Lu. borealis , displays slightly less negative values (-28.5 ± 0.7‰ for the gill and − 26.5 ± 1.2‰ in the Vm). In Tellina sp ., measured δ 15 N isotopic values are 8.30 ± 1.4‰ for the gills and 8.30 ± 1.5‰ for the Vm. In contrast, both Lucinidae show more negative signatures, − 0.71 ± 1.7‰ in the gills and 1.3 ± 1.4‰ in the Vm of Lo. orbiculatus , and − 0.03 ± 1.5‰ in the gills and 2.9 ± 1.2‰ in the Vm for Lu. borealis (Fig. 2 A). For both Lucinidae species and for both C and N, gill tissues consistently exhibit more depleted isotopic composition than the visceral mass (Wilcoxon rank-sum test, all p < 0.0001, Fig. 2 B-C). Differences were significant for δ¹³C and δ¹⁵N values between the gill and Vm in both Lo. orbiculatus and Lu. borealis (Wilcoxon rank-sum test, all p < 0.0001, Fig. 2 B-C). The heavy isotope enrichment between visceral mass and gill is around 2‰ for both nitrogen and carbon. In addition, Lucinoma borealis shows overall less negative isotopic values than Loripes orbiculatus (Wilcoxon rank-sum test, all p < 0.0001; Fig. 2 B–C). Isotopic enrichment of lucinid tissues after exposure to labelled microorganisms For both Loripes orbiculatus and Lucinoma borealis , no significant difference in δ¹³C or δ¹⁵N values was observed between values measured in T 0 2020, T 0 2023 (EC) and experimental controls (LC and LCS), in neither gill nor visceral mass ( p adj > 0.05 for all comparisons; supplementary data S1). For Lo. orbiculatus , δ¹³C values measured in gill tissues of non-exposed controls (EC-g, LC-g, LCS-g) and bacteria-fed individuals (LB-g and LBS-g) were similar ( p adj > 0.05). In contrast, individuals exposed to phytoplankton cultures (LP-g and LPS-g) showed significantly less negative δ¹³C values ( p adj < 0.05) (Fig. 3 A). In the visceral mass, δ¹³C values were generally higher than in the gill ( p adj < 0.05). For both tissues, phytoplankton-fed conditions displayed less negative δ¹³C values than bacteria-fed conditions. With regards to the effect of sulfide, no significant difference in δ¹³C values was observed between individuals exposed to sulfide and non-sulfide conditions in any given treatment (control, phytoplankton or bacteria) ( p adj > 0.05) (Fig. 3 A). In Lu. borealis , no significant differences were observed between controls (EC-g, LC-g, LCS-g) and bacteria-fed conditions (LB-g and LBS-g; p adj > 0.05). A significant enrichment in 13 C was observed in those exposed to phytoplankton LP-g (δ¹³C = − 22 .32 ± 4.64‰) and LPS-g (δ¹³C = − 19.69 ± 2.96‰) ( p adj < 0.05). In the visceral mass, δ¹³C values increased significantly across all LB-vm, LBS-vm, LP-vm and LPS-vm treatments compared to LC-vm and LCS-vm controls (Fig. 3 B). These differences were significant in multiple pairwise comparisons (p < 0.05 or p < 0.01). As for Lo. orbiculatus , the visceral mass showed clearer enrichment in 13 C compared to the gills. Overall, for both gill and visceral mass, similar trends were thus observed in the two species, with significantly less negative values for Lu. borealis compared to Lo. orbiculatus in all conditions. Overall, highest variations were observed in δ¹³C values in conditions exposed to phytoplankton, while values did not increase (gill tissue), or only slightly increase (visceral mass) when exposed to bacterial cultures ( p adj < 0.05) (Fig. 3 A-B). In both species and both tissues, addition of sulfide did not lead to significant change in δ¹³C signatures compared to the sulfide-free condition (LB versus LBS and LP versus LPS). In Lo. orbiculatus gills, δ¹⁵N values in EC-g, LC-g, LCS-g conditions were similar (Fig. 4 A). Conditions exposed to bacteria (LB-g and LBS-g) displayed comparatively higher δ¹⁵N values with LB-g = 49.42 ± 26.22‰ and LBS-g = 86.19 ± 24.81‰ ( p adj < 0.05). Individuals exposed to phytoplankton (LP-g and LPS-g) exhibited markedly higher δ¹⁵N values compared to all other groups ( p adj < 0.05). In the visceral mass, significant increases were observed in all exposed conditions. LB-vm and LBS-vm led to moderate enrichment ( p adj < 0.01). LB-vm δ¹⁵N = 84.52 ± 80.57‰ and LBS-vm δ¹⁵N = 176.22 ± 80.06‰. LB-vm and LBS-vm display very important standard deviation indicating high heterogeneity in 15 N incorporation in the visceral mass of Lo. orbiculatus . The highest δ¹⁵N values were measured in phytoplankton-exposed groups LP-vm (δ¹⁵N = 734.02 ± 213.71‰) and LPS-vm (δ¹⁵N = 968.03 ± 359.42‰), both significantly different from all other conditions ( p adj < 0.05), and also displaying high standard deviation (Fig. 4 A). In Lu. borealis , δ¹⁵N values in EC-g, LC-g, LCS-g conditions were similar (Fig. 4 B). Conditions exposed to bacteria and phytoplankton followed the same trend: higher δ¹⁵N values in LB-g (δ¹⁵N = 87.14 ± 50.93‰) and LBS-g (δ¹⁵N = 81.82 ± 21.99‰), and markedly higher values in LP-vm (δ¹⁵N = 1040.44 ± 484.02‰) and LPS-vm (δ¹⁵N = 1374.80 ± 495.37‰) ( p adj < 0.05) compared to EC-g, LC-g, LCS-g conditions (Fig. 4 B). In the visceral mass, specimens exposed to labelled bacterial culture LB-g (δ¹⁵N = 173.60 ± 53.19‰) and LBS-g (δ¹⁵N = 192.51 ± 90.71‰) were significantly enriched compared to controls ( p adj < 0.05). Phytoplankton-fed individuals LP-vm (δ¹⁵N = 2027.48 ± 419.22‰) and LPS-vm (δ¹⁵N = 2471.66 ± 742.83‰) again displayed the highest δ¹⁵N values ( p adj < 0.05), significantly different from all other conditions (Fig. 4 B). For both tissues and lucinid species, no statistical difference was observed between sulfide-free and sulfide condition for both exposed conditions (LB versus LBS and LP versus LPS). In summary, across both species, δ¹³C and δ¹⁵N values were consistently similar among EC-g, LC-g, LCS-g conditions, indicating that the experimental conditions alone did not induce major changes in stable isotope signatures. When comparing bacteria- and phytoplankton-fed conditions, signatures were consistently higher in the latter, evidencing higher enrichment in heavy isotopes when exposed to phytoplankton. Moreover, the addition of sulfide didn’t change the isotopic signature in any of the conditions. For δ¹⁵N values, both species and tissues display important standard deviation indicating heterogeneity in 15 N incorporation. The highest δ¹³C and δ¹⁵N values were always observed in the visceral mass of phytoplankton-fed individuals. Rubisco-encoding genes in symbionts metagenomes The phylogenetic tree built based on Rubisco sequences shows that rbcL amino acids sequences from symbionts of both Lucinidae cluster with form I Rubisco of other Ca . Thiodiazotropha, confirming that both symbionts harbor a form I Rubisco (Fig. 5 A). The annotated genomes of symbionts of Lo. orbiculatus and Lu. borealis both revealed a complete Rubisco operon. The genes encoding the large subunit ( rbcL ), small subunit ( rbcS ), and the activation proteins ( cbbO and cbbQ ) were located on the same strand (-), whereas the transcriptional regulator ( cbbR ) was located on the opposite strand (+). The genomic arrangement reveals a difference. In Lo. orbiculatus , all genes were present as single copy, whereas in Lu. borealis , the genes encoding activation proteins are present in two copies ( cbbO and cbbQ , Fig. 5 B–C). Amino acids sequences alignment indicates that the small Rubisco subunit differs by 13.6% between symbionts of Lo. orbiculatus and Lu. borealis , while the large subunits differ by 3.81%. Superimposition of the predicted 3D structures for both the large and small subunits in each symbiont shows high structural similarity, with the main mismatches located at the lateral termini of the proteins (Fig. 5 D). Gill metabolome and associated microbiota Analysis of metabolites composition in gills of both Lu. borealis and Lo. orbiculatus revealed a clear separation between the EC-g (T 0 ) group and all other conditions (non-exposed controls LC-g and LCS-g and exposed conditions LB-g; LBS-g; LP-g; LPS-g, Fig. 6 A-B). PERMANOVA and pairwise tests confirmed that only the EC-g condition was significatively different from all other conditions. On the other hand, other conditions didn’t show significant difference among them. The analysis of gill bacterial communities revealed a single dominant ASV in each lucinid, both ASVs matching candidate genus Ca. Thiodiazotropha (Fig. 6 C). The ASV found in Lu. borealis matched Ca . Thiodiazotropha endolucinoma, previously identified in Lucinoma borealis (GenBank accession number LT548924.1; nucleotide identity = 100%), while the ASV found in Lo. orbiculatus matched Ca . Thiodiazotropha endoloripes found in Loripes orbiculatus (GenBank accession number LT548933.1; nucleotide identity = 100%). These two ASVs differ from one another by nine base pairs. After 15 days of experiment, gill bacterial composition remained stable and identical to the EC-g group, and no differences occurred among the different exposure conditions, indicating no sign of bacterial proliferation, bacteriemia, or population switch. Minor ASVs were identified in the condition when Lucinidae were in contact of environmental bacteria (LB-g). For example, three ASVs belonging to genus Endozoicomonas occurred in low number in Lo. orbiculatus gills. In the LC-g condition, one ASV belonging to the Spirochaeta genus was found in Lo. orbiculatus gills too. Discussion Isotopic signatures support chemoautotrophic nutrition during winter low-sulfide periods Loripes orbiculatus and Lucinoma borealis exhibit δ 13 C and δ 15 N largely more negative than signatures measured in seagrass-beds associated heterotrophic organisms found in Roscoff including Tellina (this study), Ensis ensis (-18‰ for δ 13 C and 7.8‰ for δ 15 N) and Lutraria lutraria (-17,8‰ for δ 13 C and 9,8‰ for δ 15 N; Ouisse et al., 2012 ). Their δ 13 C values are on the other hand similar to those measured in bivalves harboring autotrophic sulfur-oxidizing symbionts that use a form I RubisCO like Solemya velum (Say, 1822), δ 13 C = -34 to -30‰, and Bathymodiolus thermophilus (Kenk & B. R. Wilson, 1985), δ 13 C = -30‰, (Scott et al., 2004 ) as well as other Lucinidea (Duperron et al., 2007 ). This is congruent with the RubisCO form I operon identified in both lucinid symbionts, that is to date reported in all documented lucinids (Robinson and Cavanaugh, 1995, Duperron et al., 2007 ; Ratinskaia et al., 2024). This form is composed by 8 large and 8 small subunits and induces an isotopic fractionation about − 20‰ versus source atmospheric CO 2 (δ 13 C ~ 8,5‰ for dissolved CO 2 in the ocean; O’Leary, 1988 ), leading to a final signature around ~-30‰. The visceral mass displays slightly higher δ 13 C values compared to the gill. The ~ 2‰ difference is typically found between the gill and the visceral mass in chemosymbiotic bivalves, and interpreted as evidence that carbon is fixed in the gill then transferred to the visceral mass (Hill and McQuaid, 2009 ; Riou et al., 2010 ). These findings confirm that both Lucinidae consume organic compounds produced by their chemosynthetic symbionts, and mainly rely on them even during winter. If sulfide levels are lower in winter, this could lead to slowed down metabolism and reduced carbon turn over in host tissues. Lower carbon turn-over would be consistent with our experimental results in non-fed specimens, that show no significant change in carbon signatures over 15 days. Consistent differences in δ 13 C and δ 15 N signatures found between Lo. orbiculatus and Lu. borealis (~ 2‰ difference) could be due to metabolic differences in host or symbionts physiology, or both. Indeed, Lo. orbiculatus and Lu. borealis have been phylogenetically separated for approximately 100 million years (Taylor et al., 2011 ), allowing evolutionary divergence. Besides, and despite that they co-occur, each host harbors a distinct symbiont ASV, indicating high host-symbiont fidelity as well as potential differences. The divergence levels observed in Rubisco sequences could translate into slight functional differences in the CBB cycle, and explain the difference observed in δ¹³C values, but their highly similar inferred 3D structure does not suggest this. On the other hand, the Lo. orbiculatus symbiont possesses a single copy of cbbO and cbbQ , encoding for an AAA+ ATPase and a chaperone protein, respectively, while the Lu. borealis symbiont harbors two copies of both genes. Single copy seems the standard configuration in most autotrophic bacteria (Schwedock et al., 2004 ; Sutter et al., 2015 ). These activase proteins are involved in the post-translational activation of Rubisco by inducing conformational changes and unlocking the active sites (Martinez et al., 2020). The presence of two activase systems could allow different post-translational regulation, and may thus contribute to the differences observed in δ¹³C values by modulating Rubisco activity and isotopic fractionation in a symbiont-specific manner. In plants, the presence of multiple copies of Rubisco activase genes due to events of genome duplication or tandem gene duplication events have been evidenced (Carmo-Silva et al., 2015 ) and overexpression of Rca lead to crop yield in rice (Fukayama et al., 2012 ) and may also impact the Rubisco activation in response to temperature (Rundle and Zielinski, 1991 ). In the chemolithoautotroph Acidithiobacillus ferrooxidans . multiple rubisco-encoding operons have been characterized including two sets of isoforms of CbbQ and CbbO that form hetero-oligomers which act as specific activases for two structurally diverse Rubisco forms (Heinhorst et al., 2002 ). Similar operons regulated in response to CO 2 concentrations are also present in Hydrogenovibrio marinus (Yoshizawa et al., 2004 ). Complementary analysis on the role and regulation of the Rca and Rubisco in Lucinids are needed Experimental evidence for filter-feeding ability The metabolome of both Lo. orbiculatus and Lu. borealis is altered during experiments compared to wild specimens. The metabolomic profile is a proxy for functional status (Watanabe et al., 2015 ; Zhang et al., 2024 ), so this change suggests experimental stress impacting both Lucinidae, which is not unexpected since our lab-based experiments did not replicate real life conditions. However, no mortality was observed during the 15 days of the experiment under any of the conditions. The metabolome composition was on the other hand highly similar among the different experimental conditions, suggesting that the presence of sulfide, bacteria or phytoplankton did not induce further major changes in holobionts functioning. The composition of gill microbiota did not vary either, and in that case remained similar to that of wild specimens, with overwhelming dominance of the species-specific symbiont ASV, and no sign of bacteremia or infection by any particular known pathogen. This suggests that, once acclimated to laboratory conditions, both Lucinidae species were able to maintain a functional status under all conditions, congruent with recent finding that underlined their resistance to starvation (Orgeas-Gobin et al., 2025 ). Even in the presence of abundant food sources, symbionts remained the main bacteria in both species. When exposed for 15 days to isotope-labelled phytoplanktonic cells ( C. calcitrans , ~ 3.5 µm diameter and T. lutea , ~ 6 µm), both Lucinoma borealis and Loripes orbiculatus showed increase in δ¹³C and δ¹⁵N in both gill and visceral mass. This supports the ability of both species to filter and assimilate phytoplankton-derived organic matter through their digestive system, as in other lamellibranch bivalves. The assimilation of bacteria was less evident, yet δ¹³C and δ¹⁵N values measured in tissues suggest some assimilation of bacteria-derived material, mainly in the visceral mass as expected for heterotrophy. Overall, results indicate that both lucinid species are able to filter-feed on microorganisms from different sizes classes. Interestingly, the enrichment observed in Lu. borealis was systematically higher than in Lo. orbiculatus . This may suggest interspecific differences in reliance on, or efficiency of, heterotrophic feeding. If confirmed, such differences could imply varying fitness advantages under fluctuating environmental conditions. Altogether, these findings provide experimental evidence for filter-feeding ability in lucinids. Filter-feeding ability in chemosynthetic bivalves is taxon-dependent. In Vesicomyidae, which inhabit deep-sea environments, the digestive system is highly reduced (Le Pennec and Fiala-Médioni, 1988; Le Pennec et al., 1990). Bathymodiolus and Idas mussels on the other hand possess a reduced yet functional digestive system, with functional labial palps and a stomach containing particles, with secretory and digestive cells indicating a capacity for filtering and digesting external organic matter (Le Pennec et al., 1990, 1995 , Page et al., 2013, Duperron et al., 2010). In the Lucinidae, symbiont-derived nutrition is the main energy source. Yet, their digestive system appears complete and functional (Le Pennec et al., 1990). Loripes orbiculatus reportedly hosts symbionts within its visceral mass, raising questions about their role in nutrition, infection, and symbiont reacquisition (Alcaraz et al., 2024). Deep-sea Lucinidae, Lucinoma aequizonata and Parvilucina tenuisculpta , were able to ingest radiolabeled particles, demonstrating their potential for filter-feeding (Duplessis et al., 2004 ). These authors identified a broad range of organisms in the gut of L. aequizonata , suggesting a non-selective feeding behavior. These findings are in line with our experimental results, which indicate that coastal Lucinidae can consume both bacteria and phytoplankton. Our study shows that coastal Lucinidae are also able to filter-feed. A recent study found that Lucinoma capensis , associated with mangrove sediments, exhibited C and N signatures intermediate between autotrophic and heterotrophic and evidence of diatom ingestion and assimilation, suggesting mixotrophic nutrition which was suggested ot provide dietary supplement to cope with periods of low sulfide availability (Amorim et al., 2022 ). Our results suggest that this capability also exists in Lo. orbiculatus and Lu. borealis from temperate seagrass beds, but that symbiont-derived nutrition is predominant, in natura even during low-sulfide periods such as winter. Seagrass-bed sediments are known to harbor high microbial abundances with greater diversity and metabolic activity compared to bare sediments, owing to the presence of marine plants and substantial organic-matter production, which might favor filtration as a primary feeding strategy (Mohapatra et al., 2022 ; Zhang et al., 2024 ). However, we propose that free-living microorganisms are relatively scarce and hardly accessible to lucinids in seagrass-bed sediments. Indeed, most microorganisms are either adsorbed onto plant root surfaces, embedded within extracellular polymeric substances, or attached to particles such as microplastics (Battin et al., 2016 ; Gerbersdorf et al., 2020 ; Saygin et al., 2024 ), (Zettler et al., 2013 ; Huang et al., 2020 ; Kreitsberg et al., 2021 ). Therefore, filter-feeding may not represent the optimal strategy for Lucinidae. Mixotrophy in Lucinidae: an adaptive trade-off between energetic efficiency and environmental variability? Heterotrophic metabolism is efficient in terms of ATP production but energetically costly due to the complexity of substrate acquisition, digestion, and assimilation (Spietz et al., 2019 ; Nibel et al., 2019; Gralka et al., 2023 ). Despite these costs, heterotrophs often benefit from metabolic networks that can flexibly adjust to changes in substrate, conferring a competitive edge over strictly autotrophic organisms, at least when organic carbon sources are available (Schäfer-Scherzmann & Müller, 2016). In the case of chemosymbiosis, however, the paradigm shifts. Indeed, reliance on symbiont’s autotrophy largely alleviates the cost of nutrient acquisition for hosts, as organic carbon is directly produced inside host tissues. We hypothesize that in mixotrophic organisms, autotrophy may offer the host a better energetic yield, because the symbionts carry out the most energy-demanding biochemical reactions. In this scenario, the host can save energy by outsourcing metabolic functions to its symbionts, even if autotrophy is less efficient per unit of chemical substrate (e.g., reduced sulfur compounds) than heterotrophy. However, maintaining the ability to perform facultative filter-feeding may be critical during periods of symbiont loss or low symbiont productivity. Facultative heterotrophy of animal hosts associated to autotrophic symbionts is observed in reef-building corals. Corals combine autotrophic carbon acquisition from their symbionts with heterotrophic nitrogen uptake via prey capture, highlighting the complementarity of both nutritional strategies (Rädecker et al., 2015 ). Facultative heterotrophy is also found in bivalves, with some Thyasiridae that host symbionts only facultatively (Batstone et al., 2014 ). In Lucinidae reliance on Ca . Thiodiazotropha, which can fix both carbon and nitrogen (Petersen et al., 2016 ; Ratinskaia et al., 2024) should minimize the need for filter-feeding. Maintenance of a functional digestive system in lucinid bivalves despite the establishment of chemosynthetic symbiosis 465 million years ago (Taylor et Glover, 2006) suggests that Lucinidae face selective pressure. We hypothesize that filter-feeding may be a stress-response mechanism, activated during environmentally challenging periods—such as low sulfide availability—or during physiological stress, to support both host and symbiont integrity. Future research should aim at identifying the precise environmental conditions and physiological thresholds that trigger a shift to heterotrophic nutrition in lucinid bivalves, in order to further explore the limits of their ecological niche. Declarations Acknowledgments We thank Lidwine Trouilh (GenoToul platform) for 16S rRNA sequencing and Cedric Leroux (METABOMER mass spectrometry facility) and the metabolomic platform, PtRMN, of the Muséum national d’Histoire naturelle. We also thank the Roscoff Culture Collection (RCC) and the Roscoff Centre de Ressources Biologiques Marine (CRBM). This study was funded by the Institut de l’Océan de l’Alliance Sorbonne Universités (SOG grant) and the PPR LIFEDEEPER program (National Research Agency, France 2030: ANR-22-POCE-0007). Data availability statement The 16S rRNA gene amplicon datasets generated in this study will be available in the NCBI Sequence Read Archive under BioProject: PRJNA1266712, samples accession number SAMN56450224 to SAMN56450372. Supplementary information The online version contains supplementary data available at Conflict of interest The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. Generative AI statement The author(s) declare that no Generative AI was used in the creation of this manuscript. References Amorim K, Loick-Wilde N, Yuen B, Osvatic JT, Wäge-Recchioni J, Hausmann B, Petersen JM, Fabian J, Wodarg D, Zettler ML (2022) Chemoautotrophy, symbiosis and sedimented diatoms support high biomass of benthic molluscs in the Namibian shelf. Sci Rep 12:9731. https://doi.org/10.1038/s41598-022-13571-w Batstone RT, Laurich JR, Salvo F, Dufour SC (2014) Divergent Chemosymbiosis-Related Characters in Thyasira cf. gouldi (Bivalvia: Thyasiridae). PLoS ONE 9:e92856. https://doi.org/10.1371/journal.pone.0092856 Battin TJ, Besemer K, Bengtsson MM, Romani AM, Packmann AI (2016) The ecology and biogeochemistry of stream biofilms. Nat Rev Microbiol 14:251–263. https://doi.org/10.1038/nrmicro.2016.15 Bolyen E, Rideout JR, Dillon MR, Bokulich NA, Abnet CC, Al-Ghalith GA, Alexander H, Alm EJ, Arumugam M, Asnicar F, Bai Y, Bisanz JE, Bittinger K, Brejnrod A, Brislawn CJ, Brown CT, Callahan BJ, Caraballo-Rodríguez AM, Chase J, Cope EK, Da Silva R, Diener C, Dorrestein PC, Douglas GM, Durall DM, Duvallet C, Edwardson CF, Ernst M, Estaki M, Fouquier J, Gauglitz JM, Gibbons SM, Gibson DL, Gonzalez A, Gorlick K, Guo J, Hillmann B, Holmes S, Holste H, Huttenhower C, Huttley GA, Janssen S, Jarmusch AK, Jiang L, Kaehler BD, Kang KB, Keefe CR, Keim P, Kelley ST, Knights D, Koester I, Kosciolek T, Kreps J, Langille MGI, Lee J, Ley R, Liu Y-X, Loftfield E, Lozupone C, Maher M, Marotz C, Martin BD, McDonald D, McIver LJ, Melnik AV, Metcalf JL, Morgan SC, Morton JT, Naimey AT, Navas-Molina JA, Nothias LF, Orchanian SB, Pearson T, Peoples SL, Petras D, Preuss ML, Pruesse E, Rasmussen LB, Rivers A, Robeson MS, Rosenthal P, Segata N, Shaffer M, Shiffer A, Sinha R, Song SJ, Spear JR, Swafford AD, Thompson LR, Torres PJ, Trinh P, Tripathi A, Turnbaugh PJ, Ul-Hasan S, van der Hooft JJJ, Vargas F, Vázquez-Baeza Y, Vogtmann E, von Hippel M, Walters W, Wan Y, Wang M, Warren J, Weber KC, Williamson AD, Xu ZZ, Zaneveld JR, Zhang Y, Zhu Q., Knight, R., Caporaso, J.G., 2019. Reproducible, interactive, scalable and extensible microbiome data science using QIIME 2. Nat Biotechnol 37, 852–857. https://doi.org/10.1038/s41587-019-0209-9 Breusing C, Xiao Y, Russell SL, Corbett-Detig RB, Li S, Sun J, Chen C, Lan Y, Qian P-Y, Beinart RA (2023) Ecological differences among hydrothermal vent symbioses may drive contrasting patterns of symbiont population differentiation. mSystems 8:e0028423. https://doi.org/10.1128/msystems.00284-23 Brodersen KE, Hammer KJ, Schrameyer V, Floytrup A, Rasheed MA, Ralph PJ, Kühl M, Pedersen O (2017) Sediment Resuspension and Deposition on Seagrass Leaves Impedes Internal Plant Aeration and Promotes Phytotoxic H2S Intrusion. Front Plant Sci 8:657. https://doi.org/10.3389/fpls.2017.00657 Carmo-Silva E, Scales JC, Madgwick PJ, Parry MAJ (2015) Optimizing Rubisco and its regulation for greater resource use efficiency. Plant Cell Environ 38:1817–1832. https://doi.org/10.1111/pce.12425 Caro A, Got P, Bouvy M, Troussellier M, Gros O (2009) Effects of long-term starvation on a host bivalve (Codakia orbicularis, Lucinidae) and its symbiont population. Appl Environ Microbiol 75:3304–3313. https://doi.org/10.1128/AEM.02659-08 Cheng L, Normandeau C, Bowden R, Doucett R, Gallagher B, Gillikin DP, Kumamoto Y, McKay JL, Middlestead P, Ninnemann U, Nothaft D, Dubinina EO, Quay P, Reverdin G, Shirai K, Mørkved PT, Theiling BP, van Geldern R, Wallace DWR (2019) An international intercomparison of stable carbon isotope composition measurements of dissolved inorganic carbon in seawater. Limnol Oceanography: Methods 17:200–209. https://doi.org/10.1002/lom3.10300 Conway N, Capuzzo JM, Fry B (1989) The role of endosymbiotic bacteria in the nutrition of Solemya velum: Evidence from a stable isotope analysis of endosymbionts and host. Limnol Oceanogr 34:249–255. https://doi.org/10.4319/lo.1989.34.1.0249 Cotovicz LC, Knoppers BA, Deirmendjian L, Abril G (2019) Sources and sinks of dissolved inorganic carbon in an urban tropical coastal bay revealed by δ13C-DIC signals. Estuarine. Coastal Shelf Sci 220:185–195. https://doi.org/10.1016/j.ecss.2019.02.048 Descolas-Gros C, Fontungne M (1990) Stable carbon isotope fractionation by marine phytoplankton during photosynthesis. Plant Cell Environ 13:207–218. https://doi.org/10.1111/j.1365-3040.1990.tb01305.x Dubilier N, Bergin C, Lott C (2008) Symbiotic diversity in marine animals: the art of harnessing chemosynthesis. Nat Rev Microbiol 6:725–740. https://doi.org/10.1038/nrmicro1992 Dufour SC (2005) Gill anatomy and the evolution of symbiosis in the bivalve family Thyasiridae. Biol Bull 208:200–212. https://doi.org/10.2307/3593152 Duperron S, Fiala-Médioni A, Caprais J-C, Olu K, Sibuet M (2007) Evidence for chemoautotrophic symbiosis in a Mediterranean cold seep clam (Bivalvia: Lucinidae): comparative sequence analysis of bacterial 16S rRNA, APS reductase and RubisCO genes. FEMS Microbiol Ecol 59:64–70. https://doi.org/10.1111/j.1574-6941.2006.00194.x Duperron S, Foucault P, Duval C, Goto M, Gallet A, Colas S, Marie B (2023) Multi-omics analyses from a single sample: prior metabolite extraction does not alter the 16S rRNA-based characterization of prokaryotic community in a diversity of sample types. FEMS Microbiol Lett 370:fnad125. https://doi.org/10.1093/femsle/fnad125 Duplessis MR, Dufour SC, Blankenship LE, Felbeck H, Yayanos AA (2004) Anatomical and experimental evidence for particulate feeding in Lucinoma aequizonataand Parvilucina tenuisculpta (Bivalvia: Lucinidae) from the Santa Barbara Basin. Mar Biol 145:551–561. https://doi.org/10.1007/s00227-004-1350-6 Elisabeth NH, Caro A, Césaire T, Mansot J-L, Escalas A, Sylvestre M-N, Jean-Louis P, Gros O (2014) Comparative modifications in bacterial gill-endosymbiotic populations of the two bivalves Codakia orbiculata and Lucina pensylvanica during bacterial loss and reacquisition. FEMS Microbiol Ecol 89:646–658. https://doi.org/10.1111/1574-6941.12366 Feng D, Peckmann J, Li N, Kiel S, Qiu J-W, Liang Q, Carney RS, Peng Y, Tao J, Chen D (2018) The stable isotope fingerprint of chemosymbiosis in the shell organic matrix of seep-dwelling bivalves. Chem Geol 479:241–250. https://doi.org/10.1016/j.chemgeo.2018.01.015 Fukayama H, Ueguchi C, Nishikawa K, Katoh N, Ishikawa C, Masumoto C, Hatanaka T, Misoo S (2012) Overexpression of rubisco activase decreases the photosynthetic CO2 assimilation rate by reducing rubisco content in rice leaves. Plant Cell Physiol 53:976–986. https://doi.org/10.1093/pcp/pcs042 Gerbersdorf SU, Koca K, de Beer D, Chennu A, Noss C, Risse-Buhl U, Weitere M, Eiff O, Wagner M, Aberle J, Schweikert M, Terheiden K (2020) Exploring flow-biofilm-sediment interactions: Assessment of current status and future challenges. Water Res 185:116182. https://doi.org/10.1016/j.watres.2020.116182 Gralka M, Pollak S, Cordero OX (2023) Genome content predicts the carbon catabolic preferences of heterotrophic bacteria. Nat Microbiol 8:1799–1808. https://doi.org/10.1038/s41564-023-01458-z Heinhorst S, Baker SH, Johnson DR, Davies PS, Cannon GC, Shively JM (2002) Two Copies of form I RuBisCO genes in Acidithiobacillus ferrooxidans ATCC 23270. Curr Microbiol 45:115–117. https://doi.org/10.1007/s00284-001-0094-5 Hill JM, McQuaid CD (2009) Effects of food quality on tissue-specific isotope ratios in the mussel Perna perna. Hydrobiologia 635:81–94. https://doi.org/10.1007/s10750-009-9865-y Huang Y, Xiao X, Xu C, Perianen YD, Hu J, Holmer M (2020) Seagrass beds acting as a trap of microplastics - Emerging hotspot in the coastal region? Environ Pollut 257:113450. https://doi.org/10.1016/j.envpol.2019.113450 Jumper J, Evans R, Pritzel A, Green T, Figurnov M, Ronneberger O, Tunyasuvunakool K, Bates R, Žídek A, Potapenko A, Bridgland A, Meyer C, Kohl SAA, Ballard AJ, Cowie A, Romera-Paredes B, Nikolov S, Jain R, Adler J, Back T, Petersen S, Reiman D, Clancy E, Zielinski M, Steinegger M, Pacholska M, Berghammer T, Bodenstein S, Silver D, Vinyals O, Senior AW, Kavukcuoglu K, Kohli P, Hassabis D (2021) Highly accurate protein structure prediction with AlphaFold. Nature 596:583–589. https://doi.org/10.1038/s41586-021-03819-2 Kenk VC, Wilson B (1985) A new mussel (Bivalvia, Mytilidae) from hydrothermal vents in the Galapagos Rift zone. Malacologia Koch M, Johnson C, Madden C, Pedersen O (2022) Irradiance, Water Column O2, and Tide Drive Internal O2 Dynamics and Meristem H2S Detection in the Dominant Caribbean-Tropical Atlantic Seagrass, Thalassia testudinum. Estuaries Coasts 45. https://doi.org/10.1007/s12237-022-01064-y Kreitsberg R, Raudna-Kristoffersen M, Heinlaan M, Ward R, Visnapuu M, Kisand V, Meitern R, Kotta J, Tuvikene A (2021) Seagrass beds reveal high abundance of microplastic in sediments: A case study in the Baltic Sea. Mar Pollut Bull 168:112417. https://doi.org/10.1016/j.marpolbul.2021.112417 Krueger T, Bodin J, Horwitz N, Loussert-Fonta C, Sakr A, Escrig S, Fine M, Meibom A (2018) Temperature and feeding induce tissue level changes in autotrophic and heterotrophic nutrient allocation in the coral symbiosis - A NanoSIMS study. Sci Rep 8:12710. https://doi.org/10.1038/s41598-018-31094-1 Le Pennec M, Beninger PG, Herry A (1995) Feeding and digestive adaptations of bivalve molluscs to sulphide-rich habitats. Comp Biochem Physiol Part A: Physiol 111:183–189. https://doi.org/10.1016/0300-9629(94)00211-B Le Pennec M, Donval A, Herry A (1990a) Nutritional strategies of the hydrothermal ecosystem bivalves. Progress in Oceanography. Deep-Sea Biology 24:71–80. https://doi.org/10.1016/0079-6611(90)90020-3 Lepennec M, Fialamedioni A (1988) The role of the digestive-tract of calyptogena-laubieri and calyptogena-phaseoliformis, vesicomyid bivalves of the subduction zones of japan. Oceanol Acta 11:193–199 Leroy F, Riera P, Jeanthon C, Edmond F, Leroux C, Comtet T (2012) Importance of bacterivory and preferential selection toward diatoms in larvae of Crepidula fornicata (L.) assessed by a dual stable isotope (13C, 15N) labeling approach. J Sea Res 70:23–31. https://doi.org/10.1016/j.seares.2012.02.006 von Linné C, von Linné C, Salvius L (1758) Caroli Linnaei… Systema naturae per regna tria naturae:secundum classes, ordines, genera, species, cum characteribus, differentiis, synonymis, locis. Impensis Direct. Laurentii Salvii, Holmiae. https://doi.org/10.5962/bhl.title.542 Martinez S, Grover R, Ferrier-Pagès C (2024) Unveiling the importance of heterotrophy for coral symbiosis under heat stress. mBio 15:e0196624. https://doi.org/10.1128/mbio.01966-24 McMurdie PJ, Holmes S (2013) phyloseq: an R package for reproducible interactive analysis and graphics of microbiome census data. PLoS ONE 8:e61217. https://doi.org/10.1371/journal.pone.0061217 Meng EC, Goddard TD, Pettersen EF, Couch GS, Pearson ZJ, Morris JH, Ferrin TE (2023) UCSF ChimeraX: Tools for structure building and analysis. Protein Sci 32:e4792. https://doi.org/10.1002/pro.4792 Mohapatra M, Manu S, Dash SP, Rastogi G (2022) Seagrasses and local environment control the bacterial community structure and carbon substrate utilization in brackish sediments. J Environ Manage 314:115013. https://doi.org/10.1016/j.jenvman.2022.115013 Neufeld Z, Haynes P, Garcon V, Sudre J (2002) Ocean fertilization experiments may initiate a large scale phytoplankton bloom. Geophys Res Lett - GEOPHYS RES LETT 29. https://doi.org/10.1029/2001GL013677 Niebel B, Leupold S, Heinemann M (2019) An upper limit on Gibbs energy dissipation governs cellular metabolism. Nat Metab 1:125–132. https://doi.org/10.1038/s42255-018-0006-7 O’Leary MH (1988) Carbon Isotopes in Photosynthesis: Fractionation techniques may reveal new aspects of carbon dynamics in plants. Bioscience 38:328–336. https://doi.org/10.2307/1310735 Orgeas-Gobin S, Piquet B, Marie B, Andersen AC, Tanguy A, Duperron S (2025) Symbiont retention and holobiont response under simulated sulfide deprivation in Lucinid clams from seagrass beds. Front Microbiol 16:1637201. 10.3389/fmicb.2025.1637201 PMID: 41472809; PMCID: PMC12746666 Osvatic JT, Yuen B, Kunert M, Wilkins L, Hausmann B, Girguis P, Lundin K, Taylor J, Jospin G, Petersen JM (2023) Gene loss and symbiont switching during adaptation to the deep sea in a globally distributed symbiosis. ISME J 17:453–466. https://doi.org/10.1038/s41396-022-01355-z Ouisse V, Riera P, Migné A, Leroux C, Davoult D (2012) Food web analysis in intertidal Zostera marina and Zostera noltii communities in winter and summer. Mar Biol 159:165–175. https://doi.org/10.1007/s00227-011-1796-2 Page HM, Fisher CR, Childress JJ (1990) Role of filter-feeding in the nutritional biology of a deep-sea mussel with methanotrophic symbionts. Mar Biol 104:251–257. https://doi.org/10.1007/BF01313266 Pales Espinosa E, Tanguy A, Le Panse S, Lallier F, Allam B, Boutet I (2013) Endosymbiotic bacteria in the bivalve Loripes lacteus : Localization, characterization and aspects of symbiont regulation. J Exp Mar Biol Ecol 448:327–336. https://doi.org/10.1016/j.jembe.2013.07.015 Parada AE, Needham DM, Fuhrman JA (2016) Every base matters: assessing small subunit rRNA primers for marine microbiomes with mock communities, time series and global field samples. Environ Microbiol 18:1403–1414. https://doi.org/10.1111/1462-2920.13023 Petersen JM, Kemper A, Gruber-Vodicka H, Cardini U, van der Geest M, Kleiner M, Bulgheresi S, Mußmann M, Herbold C, Seah BKB, Antony CP, Liu D, Belitz A, Weber M (2016) Chemosynthetic symbionts of marine invertebrate animals are capable of nitrogen fixation. Nat Microbiol 2:16195. https://doi.org/10.1038/nmicrobiol.2016.195 Petersen JM, Zielinski FU, Pape T, Seifert R, Moraru C, Amann R, Hourdez S, Girguis PR, Wankel SD, Barbe V, Pelletier E, Fink D, Borowski C, Bach W, Dubilier N (2011) Hydrogen is an energy source for hydrothermal vent symbioses. Nature 476:176–180. https://doi.org/10.1038/nature10325 Rädecker N, Pogoreutz C, Voolstra CR, Wiedenmann J, Wild C (2015) Nitrogen cycling in corals: the key to understanding holobiont functioning? Trends Microbiol 23:490–497. https://doi.org/10.1016/j.tim.2015.03.008 Ratinskaia L, Malavin S, Zvi-Kedem T, Vintila S, Kleiner M, Rubin-Blum M (2024a) Metabolically-versatile Ca. Thiodiazotropha symbionts of the deep-sea lucinid clam Lucinoma kazani have the genetic potential to fix nitrogen. ISME Commun 4:ycae076. https://doi.org/10.1093/ismeco/ycae076 Riou V, Duperron S, Halary S, Dehairs F, Bouillon S, Martins I, Colaço A, Serrão Santos R (2010) Variation in physiological indicators in Bathymodiolus azoricus (Bivalvia: Mytilidae) at the Menez Gwen Mid-Atlantic Ridge deep-sea hydrothermal vent site within a year. Mar Environ Res 70:264–271. https://doi.org/10.1016/j.marenvres.2010.05.008 Robinson JJ, Scott KM, Swanson ST, O’Leary MH, Horken K, Tabita FR, Cavanaugh CM (2003) Kinetic isotope effect and characterization of form II RubisCO from the chemoautotrophic endosymbionts of the hydrothermal vent tubeworm Riftia pachyptila. Limnol Oceanogr 48:48–54. https://doi.org/10.4319/lo.2003.48.1.0048 Rundle SJ, Zielinski RE (1991) Organization and expression of two tandemly oriented genes encoding ribulosebisphosphate carboxylase/oxygenase activase in barley. J Biol Chem 266:4677–4685 Saygin H, Tilkili B, Kayisoglu P, Baysal A (2024) Oxidative stress, biofilm-formation and activity responses of P. aeruginosa to microplastic-treated sediments: Effect of temperature and sediment type. Environ Res 248:118349. https://doi.org/10.1016/j.envres.2024.118349 Schrameyer V, York PH, Chartrand K, Ralph PJ, Kühl M, Brodersen KE, Rasheed MA (2018) Contrasting impacts of light reduction on sediment biogeochemistry in deep- and shallow-water tropical seagrass assemblages (Green Island, Great Barrier Reef). Mar Environ Res 136:38–47. https://doi.org/10.1016/j.marenvres.2018.02.008 Schuchmann K, Müller V (2016) Energetics and Application of Heterotrophy in Acetogenic Bacteria. Appl Environ Microbiol 82:4056–4069. https://doi.org/10.1128/AEM.00882-16 Schwedock J, Harmer TL, Scott KM, Hektor HJ, Seitz AP, Fontana MC, Distel DL, Cavanaugh CM (2004) Characterization and expression of genes from the RubisCO gene cluster of the chemoautotrophic symbiont of Solemya velum: cbbLSQO. Arch Microbiol 182:18–29. https://doi.org/10.1007/s00203-004-0689-x Scott KM, Schwedock J, Schrag DP, Cavanaugh CM (2004) Influence of form IA RubisCO and environmental dissolved inorganic carbon on the delta13C of the clam-chemoautotroph symbiosis Solemya velum. Environ Microbiol 6:1210–1219. https://doi.org/10.1111/j.1462-2920.2004.00642.x Sogin EM, Leisch N, Dubilier N (2020) Chemosynthetic symbioses. Curr Biol 30:R1137–R1142. https://doi.org/10.1016/j.cub.2020.07.050 Spietz RL, Lundeen RA, Zhao X, Nicastro D, Ingalls AE, Morris RM (2019) Heterotrophic carbon metabolism and energy acquisition in Candidatus Thioglobus singularis strain PS1, a member of the SUP05 clade of marine Gammaproteobacteria. Environ Microbiol 21:2391–2401. https://doi.org/10.1111/1462-2920.14623 Sutter M, Roberts EW, Gonzalez RC, Bates C, Dawoud S, Landry K, Cannon GC, Heinhorst S, Kerfeld CA (2015) Structural Characterization of a Newly Identified Component of α-Carboxysomes: The AAA+ Domain Protein CsoCbbQ. Sci Rep 5:16243. https://doi.org/10.1038/srep16243 Tamura K, Stecher G, Kumar S (2021) Mol Biol Evol 38:3022–3027. https://doi.org/10.1093/molbev/msab120 . MEGA11: Molecular Evolutionary Genetics Analysis Version 11 Taylor JD, Glover EA (2000) Functional anatomy, chemosymbiosis and evolution of the Lucinidae. Geological Society, London, Special Publications 177, 207–225. https://doi.org/10.1144/GSL.SP.2000.177.01.12 Taylor JD, Glover EA, Smith L, Dyal P, Williams ST (2011) Molecular phylogeny and classification of the chemosymbiotic bivalve family Lucinidae (Mollusca: Bivalvia). Zool J Linn Soc 163:15–49. https://doi.org/10.1111/j.1096-3642.2011.00700.x Thorsen S, Kristensen E, Valdemarsen T, Flindt M, Organo Quintana C, Holmer M (2019) Fertilizer-derived N in opportunistic macroalgae after flooding of agricultural land. Mar Ecol Prog Ser 616. https://doi.org/10.3354/meps12927 Thubaut J, Corbari L, Gros O, Duperron S, Couloux A, Samadi S (2013a) Integrative biology of Idas iwaotakii (Habe, 1958), a model species associated with sunken organic substrates. PLoS ONE 8:e69680. https://doi.org/10.1371/journal.pone.0069680 Tsai Y-CC, Ye F, Liew L, Liu D, Bhushan S, Gao Y-G, Mueller-Cajar O (2020) Insights into the mechanism and regulation of the CbbQO-type Rubisco activase, a MoxR AAA+ ATPase. Proc Natl Acad Sci U S A 117:381–387. https://doi.org/10.1073/pnas.1911123117 Watanabe M, Meyer KA, Jackson TM, Schock TB, Johnson WE, Bearden DW (2015) Application of NMR-based metabolomics for environmental assessment in the Great Lakes using zebra mussel (Dreissena polymorpha). Metabolomics 11:1302–1315. https://doi.org/10.1007/s11306-015-0789-4 Yilmaz P, Parfrey LW, Yarza P, Gerken J, Pruesse E, Quast C, Schweer T, Peplies J, Ludwig W, Glöckner FO (2014) The SILVA and All-species Living Tree Project (LTP) taxonomic frameworks. Nucleic Acids Res 42:D643–648. https://doi.org/10.1093/nar/gkt1209 Yoshizawa Y, Toyoda K, Arai H, Ishii M, Igarashi Y (2004) CO2-responsive expression and gene organization of three ribulose-1,5-bisphosphate carboxylase/oxygenase enzymes and carboxysomes in Hydrogenovibrio marinus strain MH-110. J Bacteriol 186:5685–5691. https://doi.org/10.1128/JB.186.17.5685-5691.2004 Zettler ER, Mincer TJ, Amaral-Zettler LA (2013) Life in the plastisphere: microbial communities on plastic marine debris. Environ Sci Technol 47:7137–7146. https://doi.org/10.1021/es401288x Zhang Y, Wang H, Liu S, Kong X, Chang L, Zhao L, Bao Z, Hu X (2024) Multi-tissue metabolomic profiling reveals the crucial metabolites and pathways associated with scallop growth. BMC Genomics 25:1091. https://doi.org/10.1186/s12864-024-11016-4 Zhang Y, Wang Q, Yao Y, Tan F, Jiang L, Shi W, Yang W, Liu J (2024) Bacterial Communities in Zostera marina Seagrass Beds of Northern China. Water 16:935. https://doi.org/10.3390/w16070935 Supplementary Files Supplementarydata.docx Cite Share Download PDF Status: Under Review Version 1 posted Reviewers agreed at journal 28 Apr, 2026 Reviewers invited by journal 27 Apr, 2026 Editor assigned by journal 20 Apr, 2026 First submitted to journal 08 Apr, 2026 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-9360400","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":630714630,"identity":"1de8241d-e030-473c-a94b-25969c921ccb","order_by":0,"name":"Samuel Orgeas-Gobin","email":"","orcid":"","institution":"Sorbonne University Pierre and Marie Curie Campus: Sorbonne Universite Campus Pierre et Marie Curie","correspondingAuthor":false,"prefix":"","firstName":"Samuel","middleName":"","lastName":"Orgeas-Gobin","suffix":""},{"id":630714631,"identity":"ba83ac4f-14d6-41bd-93a3-230b54c4cf3a","order_by":1,"name":"Alexandre Nguyen-tiet","email":"","orcid":"","institution":"Technical University of Denmark: Danmarks Tekniske Universitet","correspondingAuthor":false,"prefix":"","firstName":"Alexandre","middleName":"","lastName":"Nguyen-tiet","suffix":""},{"id":630714632,"identity":"b5e86688-5078-4a47-b171-465265ed9a1d","order_by":2,"name":"Lucie Blondel","email":"","orcid":"","institution":"Sorbonne Université: Sorbonne Universite","correspondingAuthor":false,"prefix":"","firstName":"Lucie","middleName":"","lastName":"Blondel","suffix":""},{"id":630714633,"identity":"2fa804dc-1856-45e3-b941-fc71715dabb6","order_by":3,"name":"Benjamin Marie","email":"","orcid":"","institution":"Muséum d'histoire naturelle: Museum d'histoire naturelle","correspondingAuthor":false,"prefix":"","firstName":"Benjamin","middleName":"","lastName":"Marie","suffix":""},{"id":630714634,"identity":"a7a06277-d743-4651-a659-ada19df50615","order_by":4,"name":"Pascal Riera","email":"","orcid":"","institution":"Sorbonne Université Campus Pierre et Marie Curie: Sorbonne Universite Campus Pierre et Marie Curie","correspondingAuthor":false,"prefix":"","firstName":"Pascal","middleName":"","lastName":"Riera","suffix":""},{"id":630714635,"identity":"4f2d9776-24d2-4168-a717-6e9996b353e3","order_by":5,"name":"Sébastien Duperron","email":"","orcid":"","institution":"National Museum for Natural History: Museum National d'Histoire Naturelle","correspondingAuthor":false,"prefix":"","firstName":"Sébastien","middleName":"","lastName":"Duperron","suffix":""},{"id":630714636,"identity":"307cf31e-8d42-4606-ade4-25bf625d7af6","order_by":6,"name":"Arnaud Tanguy","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAABBUlEQVRIiWNgGAWjYNACNhjDQEIORB14QIQWCQijwMYYrCWBeC0f0hIbQDQ+Lfyzzx78zFPGUMc/u/2aNI/B4fT5YYcfAm2xk9NtwK5F4lxesjTPOQYJiTtnykBacjfeTjMAakk2NjuAw5ozPAbSvG1Ah93ISYNomZ0A0nIgcRsOLfJneIx/g7TIQ7WkG85O/4BXi8EZHjOwLQY30o8BtaQlyEvn4LfFEKjFcs45CcmNN3KYLecY2BhukM4pOJBggNsvckCH3XhTZsMvdyP94Y03fyTk5Wenb/7wocJODqf3IQAULTwGTDwgp4JVGuBVDgPsDxh/ACn5BqJUj4JRMApGwQgCAOHPXG718d66AAAAAElFTkSuQmCC","orcid":"https://orcid.org/0000-0002-9670-2693","institution":"Sorbonne University: Sorbonne Universite","correspondingAuthor":true,"prefix":"","firstName":"Arnaud","middleName":"","lastName":"Tanguy","suffix":""}],"badges":[],"createdAt":"2026-04-08 18:48:50","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-9360400/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-9360400/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":108596887,"identity":"76035486-4c16-4253-ae52-74dc7d926a93","added_by":"auto","created_at":"2026-05-06 10:48:38","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":132315,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eExperimental design for the 15 days feeding experiment.\u003c/strong\u003e EC: (Environmental T\u003csub\u003e0 \u003c/sub\u003econtrol). This control includes seven individuals per species directly sampled from the seagrass-bed in February 2023. LC: (Lucinidae control), Lucinidae only. LS: (Sulfur control), Lucinidae exposed to a sulfur source. LB and LP: (respectively bacteria or phytoplankton condition 1), Lucinidae exposed to isotope-labelled bacteria or phytoplankton (\u003csup\u003e13\u003c/sup\u003eC + \u003csup\u003e15\u003c/sup\u003eN). LBS and LPS: (respectively bacteria or phytoplankton condition 2) Lucinidae exposed to isotope-labelled bacteria or phytoplankton (\u003csup\u003e13\u003c/sup\u003eC + \u003csup\u003e15\u003c/sup\u003eN), in the presence of a sulfur source (Na\u003csub\u003e2\u003c/sub\u003eS).\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-9360400/v1/51413b97eeca087567c2f8aa.png"},{"id":108596888,"identity":"799f571b-3d17-4d34-ace1-c4db8392281e","added_by":"auto","created_at":"2026-05-06 10:48:38","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":96841,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eIsotopic signature of gill and visceral mass (Vm) of Lucinidae and \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eTellina\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e bivalves sampled in 2020.\u003c/strong\u003e (A) Biplot represents natural δ\u003csup\u003e15\u003c/sup\u003eN and δ\u003csup\u003e13\u003c/sup\u003eC signatures. \u003cem\u003eLo. orbiculatus\u003c/em\u003e Vm samples are displayed in dark blue and gill in light blue (n= 30). \u003cem\u003eLu. borealis\u003c/em\u003e Vm samples are displayed in brown and gill in light brown (n= 10). \u003cem\u003eTellina\u003c/em\u003e sp. Vm and gill samples are represented in grey and black, respectively (n= 3). (B-C) Boxplots represents δ¹³C and δ\u003csup\u003e15\u003c/sup\u003eN values in both Lucinidae and tissues (\u003cem\u003eTellina\u003c/em\u003e sp. not shown). Significant pairwise Wilcoxon rank-sum tests are displayed with asterisks (p \u0026lt; 0.05).\u003c/p\u003e","description":"","filename":"2.png","url":"https://assets-eu.researchsquare.com/files/rs-9360400/v1/e712fe2bb97ed5b4d9f91267.png"},{"id":108805076,"identity":"7a630994-3f27-435f-b0f6-688ed83fe3d1","added_by":"auto","created_at":"2026-05-08 15:24:42","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":64995,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eLucinidae δ¹³C isotopic signatures in the different treatments.\u003c/strong\u003e (A) \u003cem\u003eLo. orbiculatus\u003c/em\u003e gills and visceral mass (Vm) δ¹³C isotopic signatures\u003cstrong\u003e \u003c/strong\u003eafter 15 days of exposure to labelled microorganisms versus controls. Controls and experimental conditions as noted as follows: bacterial communities without sulfide (LB) or with sulfide (LBS); phytoplankton without sulfide (LP) or with sulfide (LPS); T\u003csub\u003e0\u003c/sub\u003e environmental control (EC); experimental control without sulfide (LC) or with sulfide (LCS). Significant changes are indicated with a black bar, and p-values are shown as p\u003csub\u003eadj \u003c/sub\u003e\u0026lt; 0.05 (*) or p\u003csub\u003eadj \u003c/sub\u003e\u0026lt; 0.01 (**). A significant difference between controls and experimental conditions is confirmed only when the experimental condition differs significantly from all three controls (EC, LC and LCS). n=5 individuals per treatment; total n= 35 individuals per species.\u003c/p\u003e","description":"","filename":"3.png","url":"https://assets-eu.researchsquare.com/files/rs-9360400/v1/afa9fa9497659d518ff3bd63.png"},{"id":108596889,"identity":"79b25a23-b934-4972-83ac-2bae94e99255","added_by":"auto","created_at":"2026-05-06 10:48:38","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":56403,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eLucinidae δ¹\u003c/strong\u003e\u003csup\u003e\u003cstrong\u003e5\u003c/strong\u003e\u003c/sup\u003e\u003cstrong\u003eN isotopic signatures in the different treatments.\u003c/strong\u003e (A) \u003cem\u003eLo. orbiculatus\u003c/em\u003e gills and Vm δ¹\u003csup\u003e5\u003c/sup\u003eN\u003cstrong\u003e \u003c/strong\u003eisotopic signatures\u003cstrong\u003e \u003c/strong\u003eafter 15 days of exposure to labelled microorganisms versus controls. Controls and experimental conditions as noted as follow: bacterial communities without sulfide (LB) or with sulfide (LBS); phytoplankton without sulfide (LP) or with sulfide (LPS); T\u003csub\u003e0\u003c/sub\u003e environmental control (EC); Lucinidae experimental control without sulfide (LC) or with sulfide (LCS). Significant changes are indicated with a black bar, and p-values are shown as p\u003csub\u003eadj \u003c/sub\u003e\u0026lt; 0.05 (*) or p\u003csub\u003eadj \u003c/sub\u003e\u0026lt; 0.01 (**). A significant difference between controls and experimental conditions is confirmed only when the experimental condition differs significantly from all three controls (EC, LC and LCS). n=5 individuals per treatment; total n= 35 individuals per species.\u003c/p\u003e","description":"","filename":"4.png","url":"https://assets-eu.researchsquare.com/files/rs-9360400/v1/80f1ba4fcf3d9db75c43ffa1.png"},{"id":108804944,"identity":"742aeea2-8317-4341-a99d-844433919309","added_by":"auto","created_at":"2026-05-08 15:24:19","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":117582,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eComparative analysis of Rubisco in \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eLucinoma borealis\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e and \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eLoripes orbiculatus\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e symbionts. \u003c/strong\u003e(A) Rooted tree based on Rubisco multiple alignments of rbcL amino acids sequences using MUSCLE and maximum likelihood tree. Lucinidae’ symbionts are showed in bold on the tree, bootstrap values (1000 replicates) are provided at nodes, and branch lengths on respective branches. (B)\u003cstrong\u003e \u003c/strong\u003eThe\u003cstrong\u003e \u003c/strong\u003eRubisco operon of the \u003cem\u003eLo. orbiculatus\u003c/em\u003e symbiont. (C) \u003cem\u003eLu. borealis\u003c/em\u003e symbiont\u003cstrong\u003e \u003c/strong\u003eRubisco operon. (D) Superimposition of the predicted 3D structures of rbcL and rbcS subunits; \u003cem\u003eLo. orbiculatus\u003c/em\u003ein blue, \u003cem\u003eLu. borealis\u003c/em\u003e’ in brown. Amino acids sequences accession number are available in supplementary_table_S1.\u003c/p\u003e","description":"","filename":"5.png","url":"https://assets-eu.researchsquare.com/files/rs-9360400/v1/b1bcef9858513b1c9a730efa.png"},{"id":108596891,"identity":"c56f6867-c796-43dc-8a5f-33fdb5d45b28","added_by":"auto","created_at":"2026-05-06 10:48:38","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":114546,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eLucinidae associated gill metabolome and microbiota composition.\u003c/strong\u003e (A) Principal Coordinates Analysis (PCoA) of \u003cem\u003eLo. orbiculatus\u003c/em\u003e gill metabolome composition based on euclidean distance. (B) PCoA of \u003cem\u003eLu. borealis\u003c/em\u003e gill metabolome composition. Colored polygons indicate the convex hulls of the points for each group, representing the minimal convex area enclosing all observations. (C) Relative abundance of bacterial 16S metabarcoding reads matching bacterial genera in the gills; \u003cem\u003eCa.\u003c/em\u003e Thiodiazotropha endoloripes ASV is in blue; \u003cem\u003eCa.\u003c/em\u003e Thiodiazotropha endolucinoma ASV is in brown. Other genera (\u0026lt; 0.5%) are grouped in “other”. Each bar represents the mean relative abundance of ASVs across the six specimens for that condition.\u003c/p\u003e","description":"","filename":"6.png","url":"https://assets-eu.researchsquare.com/files/rs-9360400/v1/dc026cb9753e8d8241b27b6c.png"},{"id":108809866,"identity":"c27ec9d7-0825-4f95-bf9b-fda8cbf0001b","added_by":"auto","created_at":"2026-05-08 15:55:58","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":880723,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-9360400/v1/7a528021-5eab-4de0-82e5-82b9efb328d8.pdf"},{"id":108805042,"identity":"fd514ae7-5fb5-43ba-9e3d-724d475c0f97","added_by":"auto","created_at":"2026-05-08 15:24:36","extension":"docx","order_by":4,"title":"","display":"","copyAsset":false,"role":"supplement","size":844525,"visible":true,"origin":"","legend":"","description":"","filename":"Supplementarydata.docx","url":"https://assets-eu.researchsquare.com/files/rs-9360400/v1/a4dd10e78cdd44de68c4833d.docx"}],"financialInterests":"","formattedTitle":"Experimental evidence of mixotrophy in seagrass-associated Lucinidae in the absence of sulfide","fulltext":[{"header":"Introduction","content":"\u003cp\u003eLucinidae bivalves harbor chemosynthetic bacterial symbionts and inhabit seagrass beds and mangroves habitats, among others, where oxic-anoxic interfaces occur near the rhizosphere (Dubilier et al, \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e2008\u003c/span\u003e, Osvatic et al., \u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). Seagrass beds are characterized by high organic matter inputs from plant litter, and strong biogeochemical fluctuations. In the underlying oxygen-depleted sediments, this organic matter is degraded by sulfate-reducing bacteria, which use sulfate as an electron acceptor and produce H₂S as a by-product (J\u0026oslash;rgensen, 1977; Boudreau \u0026amp; Westrich, 1984). Reduced sulfur compounds, notably H₂S, serves as a substrate for the chemosynthetic Gammaproteobacteria occurring in the gill bacteriocytes of Lucinidae (Dando et al., 1986; Johnson \u0026amp; Fern\u0026aacute;ndez, 2001; Pales Espinosa et al., \u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e2013\u003c/span\u003e). Plant roots also release oxygen into the surrounding sediment (Sand-Jensen et al., 2005; Heide et al., 2012; Sanmart\u0026iacute; et al., 2018), supporting the activity of lucinids and symbionts and promoting plant growth and ecosystem stability. Besides sulfur oxidation, genomes of some symbionts also encode nitrogenase, enabling biological nitrogen fixation, which may benefit both the host and the plant by alleviating the nitrogen limitation inherent to seagrass beds (Petersen et al., 2017; K\u0026ouml;nig et al., 2017; Cardini et al., 2019). Because sulfide is toxic to both animals and plants, Lucinidae symbioses also contribute to detoxification of the host environment and rhizosphere. For this reason, authors have proposed the hypothesis of a tripartite symbiosis between seagrass-bed, Lucinidae and Lucinidae-associated symbionts (Distel, 1998; Van der Geest, 2013).\u003c/p\u003e \u003cp\u003eSeagrass beds-associated Lucinidae are considered as primarily relying on symbiont-derived organic compounds. However, environmental fluctuations (e.g., tides, sunlight, temperature) and anthropogenic pressures (e.g., global changes, eutrophication, sediment resuspension) cause variations in H₂S availability, resulting in alternating low- and high-sulfide periods (Kock et al., 2022). During winter, decreased temperature and light reduce both microbial and photosynthetic activity, lowering sulfide production. This raises questions about the persistence of chemosynthetic nutrition and the fate of symbionts (Brodersen et al., \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e2017\u003c/span\u003e; Shrameyer et al., 2018). Conversely, in nutrient-rich agricultural regions, fertilizer runoffs promote algal and microbial blooms, increasing organic matter degradation and H₂S production (Neufeld et al., \u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e2002\u003c/span\u003e; Thorsen et al., \u003cspan citationid=\"CR67\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). These shifts can occur rapidly, requiring some level of plasticity to deal with changes.\u003c/p\u003e \u003cp\u003eSeveral studies have shown that mangrove- and seagrass-associated Lucinidae can survive extended periods of sulfide starvation (Caro et al., \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e2009\u003c/span\u003e; Elisabeth et al., \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e2014\u003c/span\u003e; Orgeas-Gobin et al., \u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e2025\u003c/span\u003e), displaying symbiont density decline. To compensate, Lucinidae may supplement their nutrition through heterotrophy. For example, gut content analyses of \u003cem\u003eLucinoma borealis\u003c/em\u003e revealed ingested diatoms, suggesting facultative heterotrophy (Dando et al., 1986). In \u003cem\u003eLoripes orbiculatus\u003c/em\u003e, seasonal changes in the ratio of bacteriocytes to mucocytes in gill tissues also suggest potential shifts in nutritional mode (Roques et al., 2020).\u003c/p\u003e \u003cp\u003eIn this study, we test the hypothesis that coastal lucinids are mixotrophic, supplementing autotrophy with filter-feeding during low sulfide periods. For this, specimens of \u003cem\u003eLoripes orbiculatus\u003c/em\u003e and \u003cem\u003eLucinoma borealis\u003c/em\u003e were collected during winter to assess their natural C and N isotopic signatures during a presumed low-sulfide period. To test their potential for filter-feeding, specimens were then experimentally exposed to \u003csup\u003e13\u003c/sup\u003eC- and \u003csup\u003e15\u003c/sup\u003eN-labeled cultures of either bacteria or larger phytoplankton, representing different types of potential prey, with or without a sulfide source. Potential shifts in symbiont community composition were investigated by analyzing bacterial community composition using 16S rRNA metabarcoding, and changes in holobionts functional profile were explored using non-targeted metabolome analysis. Results from both lucinid species, as well as the organization of symbionts Rubisco-encoding genes, were compared. To our knowledge, this study provides the first experimental evidence of a filter-feeding strategy in lucinids.\u003c/p\u003e"},{"header":"Material and methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eLucinidae Collection\u003c/h2\u003e \u003cp\u003eTwo groups of Lucinidae were collected at different periods for two distinct analyses: the natural isotopic signature analysis (2020 Lucinidae group) and the feeding experiment (2023 Lucinidae group). Specimens were collected in the sublittoral area adjacent to a \u003cem\u003eZostera marina\u003c/em\u003e seagrass bed near the Roscoff Biological Station (Roscoff, France; 48.7314 N, -4.0024 W; sediment depth: 20 cm). For the natural isotopic signature measurement, a total of 30 \u003cem\u003eLo. orbiculatus\u003c/em\u003e, 10 \u003cem\u003eLu. borealis\u003c/em\u003e and 3 \u003cem\u003eTellina sp.\u003c/em\u003e were collected. \u003cem\u003eTellina sp.\u003c/em\u003e is a non-symbiotic heterotrophic bivalve that lives in sympatry in seagrass-beds (heterotrophic control). Upon collection, clams were dissected under sterile conditions as follows: hemibranchs and visceral mass were flash-frozen separately in liquid nitrogen and stored at -80\u0026deg;C until mass spectrometry (MS) analysis. For the feeding experiment, 120 \u003cem\u003eLo. orbiculatus\u003c/em\u003e and 120 \u003cem\u003eLu. borealis\u003c/em\u003e were collected in February 2023. Directly upon collection, seven individuals per species were dissected under sterile conditions (T₀ environmental samples, natural isotopic reference samples) and compared with those collected in February 2020. The remaining individuals were maintained in aquaria at the Roscoff Marine Biological Research Center (CRBM). During feeding experiments, all Lucinidae were dissected as follows: the two hemibranchs and the remaining visceral mass were flash-frozen separately in liquid nitrogen and stored at \u0026minus;\u0026thinsp;80\u0026deg;C. One hemibranch and the visceral mass of each individual were used to assess isotopic composition via mass spectrometry, while the second hemibranch was used for sequential metabolite and DNA extraction for metabolomic and metabarcoding analysis.\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eMicroorganism cultivation and stable isotopic labeling\u003c/h3\u003e\n\u003cp\u003eFor the feeding experiment, two cultures of microorganisms were produced: one consists of environmental bacteria isolated from seagrass bed sediments, and the second is a mixture of two phytoplankton strains, \u003cem\u003eTisochrysis lutea\u003c/em\u003e RCC\u0026sup1;\u0026sup3;⁴⁹ and \u003cem\u003eChaetoceros calcitrans\u003c/em\u003e RCC\u0026sup1;⁸⁰⁹, obtained from the Roscoff Culture Collection (RCC). Bacteria were directly sampled from seagrass bed sediments at a depth of 20 cm. The sediments were inoculated into Marine Broth 2216 medium (Millipore, Burlington, MA, USA), and incubated for seven days at 14\u0026deg;C in the dark until reaching the stationary phase. Phytoplankton strains were cultured separately under sterile conditions (24 h light, 20\u0026deg;C) in 10 L oxygenated flasks. \u003cem\u003eT. lutea\u003c/em\u003e was grown in sterile seawater supplemented with 1 mL/L of Conway medium (Lananan et al., 2013) and bubbled with CO₂ until reaching the stationary phase (seven days). \u003cem\u003eC. calcitrans\u003c/em\u003e was cultivated in a mixture of sterile seawater (2/3) and freshwater (1/3), also supplemented with 1 mL/L of Conway medium and bubbled with CO₂, reaching the stationary phase after five days. Both phytoplankton strains were then mixed together. After microorganism cultivation, two distinct feeding mixture were obtained, the environmental bacteria and the phytoplankton cultures. Once both microorganism cultures reached a density of 10⁶ CFU/mL, 5 L of each were harvested and stored at 4\u0026deg;C to halt further growth. A subsample of each unlabelled culture was used to determine the baseline isotopic signatures (control samples). The remaining cultures were then labelled by adding 500 mL of a 4 mM \u0026sup1;\u0026sup3;C solution (NaH\u0026sup1;\u0026sup3;CO₃; 99% \u0026sup1;\u0026sup3;C) and 500 mL of a 1 g\u0026middot;L⁻\u0026sup1; \u0026sup1;⁵N solution (\u0026sup1;⁵NH₄Cl; 98% \u0026sup1;⁵N; Eurisotop, Saarbr\u0026uuml;cken, Germany), and incubated for four days. These concentrations were selected based on previous isotopic labelling experiments on microalgae (Leroy et al., \u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e2012\u003c/span\u003e). Following labelling, cultures were centrifuged twice (15 minutes, 7000 g, 20\u0026deg;C) to remove residual non-incorporated isotopes and terminate further incorporation. The resulting pellets were resuspended in sterile seawater and stored at 4\u0026deg;C until further use. A fraction of each labelled culture was preserved to assess the post-labelling isotopic enrichment. Elemental analysis shows that microorganisms sources have been well enriched:\u003cem\u003eT. lutea\u003c/em\u003e δ\u003csup\u003e13\u003c/sup\u003eC\u0026thinsp;=\u0026thinsp;598.8\u0026permil; and δ\u003csup\u003e15\u003c/sup\u003eN\u0026thinsp;=\u0026thinsp;195,948.7\u0026permil;, \u003cem\u003eC. calcitrans\u003c/em\u003e δ\u003csup\u003e13\u003c/sup\u003eC\u0026thinsp;=\u0026thinsp;394.1\u0026permil; and δ\u003csup\u003e15\u003c/sup\u003eN\u0026thinsp;=\u0026thinsp;223,535.3\u0026permil;, environmental bacteria δ\u003csup\u003e13\u003c/sup\u003eC\u0026thinsp;=\u0026thinsp;14,494.8\u0026permil; and δ\u003csup\u003e15\u003c/sup\u003eN\u0026thinsp;=\u0026thinsp;525.3\u0026permil;.\u003c/p\u003e\n\u003ch3\u003eFeeding experiment\u003c/h3\u003e\n\u003cp\u003eSix 3-L aquaria were set up room, each containing 2 L of 0.22 \u0026micro;m-filtered seawater (water temperature\u0026thinsp;=\u0026thinsp;14\u0026deg;C, salinity\u0026thinsp;=\u0026thinsp;35 PSU, room temperature\u0026thinsp;=\u0026thinsp;20\u0026deg;C). Aquarium conditions are summarized in Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eFor experimental conditions, twenty \u003cem\u003eLo. orbiculatus\u003c/em\u003e and twenty \u003cem\u003eLu. borealis\u003c/em\u003e individuals were present in each aquarium. In aquaria supplemented with labelled microorganisms, 300 mL of labelled culture (10⁶ CFU/mL) were added. For aquaria receiving sulfide, two dialysis bags, each containing 1.5 g of Na₂S, were placed centrally among the bivalves to ensure gradual sulfide release. Sampling was performed at days 7 and 15 (T7 and T15). At each time point, seven individuals per species were collected for isotopic analysis via mass spectrometry. Half of the aquarium water was replaced daily, and the dialysis bags containing Na\u003csub\u003e2\u003c/sub\u003eS were renewed. Every two days, 200 mL of labelled microorganisms (2 *10\u003csup\u003e8\u003c/sup\u003e CFU) culture was added. To avoid bias in isotopic analysis, individuals were placed in sterile seawater for 48 h post-experimentation to eliminate non-incorporated isotopes prior to dissection.\u003c/p\u003e\n\u003ch3\u003eMass spectrometry analysis\u003c/h3\u003e\n\u003cp\u003eTissues were flash-frozen in liquid nitrogen and stored at -80\u0026deg;C. To prevent isotopic cross-contamination, natural (EC, LC and LS) and enriched (LB, LBS, LP and LPS) samples were handled in separate laboratories using dedicated equipment. Frozen samples were placed into open 2-mL Eppendorf tubes and dried in an oven at 50\u0026deg;C for 48 hours. Dried, tissues were ground directly in the tubes using a sterile spatula. Between 0.80 and 1.20 mg of powdered tissue was weighed into tin capsules, sealed, and stored until mass spectrometry analysis. Carbon and nitrogen isotopic ratios were determined using a CHN elemental analyzer (ThermoFinnigan 1112 Series) coupled to an isotope ratio mass spectrometer (ThermoFinnigan MAT Deltaplus) via a Finnigan Con-Flo III interface. Data are expressed in δ (\u0026permil;), representing the deviation relative to international reference standards: Vienna Pee Dee Belemnite for δ\u0026sup1;\u0026sup3;C and atmospheric N₂ for δ\u0026sup1;⁵N. Laboratory standards were calibrated using certified reference materials: NBS19 for carbon and IAEA-N3 for nitrogen. The standard deviations of repeated measurements of δ\u0026sup1;\u0026sup3;C and δ\u0026sup1;⁵N in lab standards were 0.10\u0026permil; relative to V-PDB and 0.05\u0026permil; relative to atmospheric nitrogen, respectively. Statistical analyses were conducted in R (R version 4.4.1, 2024-06-14 ucrt, Posit, PBC). Since the enriched isotopic data did not meet the assumptions of normality and homogeneity of variances (as assessed by the Shapiro-Wilk and Levene\u0026rsquo;s tests, respectively), we applied an exact permutation test (Fisher-Pitman), implemented in the coin package. Multiple comparisons were adjusted using the Benjamini-Hochberg method. This test is recommended for small sample sizes and datasets with high dispersion, as observed in our case.\u003c/p\u003e\n\u003ch3\u003eSequential extraction of metabolites and DNA\u003c/h3\u003e\n\u003cp\u003eMetabolite and DNA extractions were performed to analyze the metabolome and microbiome of the same individual following the protocol described in Duperron et al, \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e2023\u003c/span\u003e. Briefly, samples were suspended in 200 \u0026micro;L of cold UHPLC-grade methanol-water (75:25%), homogenized mechanically (GLH850 OMNI, 25,000 rpm, 30 s), and sonicated (Sonics Vibra-Cell VCX 130, 60% amplitude, 30 s) on ice. Supernatants collected post-centrifugation (15,300 g, 4\u0026deg;C, 10 min) were stored in amber vials at -20\u0026deg;C for LC/MS analysis (see below). DNA was then extracted from pellets using the QIAGEN PowerLyzer PowerSoil DNA Kit (Hilde, Germany) following the manufacturer's instructions and using FastPrep 5G disruption (5\u0026times;30 s, 8 m/s). An extraction blank was included. Metabolites from hemibranchs of five specimens of \u003cem\u003eLo. orbiculatus\u003c/em\u003e and \u003cem\u003eLu. borealis\u003c/em\u003e were analyzed using ultra-high-performance liquid chromatography (UHPLC; ELUTE, Bruker) coupled with high-resolution mass spectrometry (ESI-Qq-TOF Compact, Bruker). Metabolomic analyses were performed with MetaboAnalyst 6.0, (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://www.metaboanalyst.ca/\u003c/span\u003e\u003cspan address=\"https://www.metaboanalyst.ca/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003cb\u003e)\u003c/b\u003e using standard parameters for metabolomics data (Pang et al.,2024). Statistical analyses were conducted in R (R version 4.4.1, 2024-06-14 ucrt, Posit, PBC) using PERMANOVA and pairwise-test using euclidean distance with FDR correction.\u003c/p\u003e \u003cp\u003eFollowing DNA extraction, a PCR1 was performed to amplify a fragment of bacterial 16S rRNA-encoding genes on all samples using primers 341F (5\u0026prime;-CCTACGGGNGGCWGCAG\u0026thinsp;\u0026minus;\u0026thinsp;3\u0026prime;) and 806R (5\u0026prime;-GGACTACVSGGGTATCTAAT\u0026thinsp;\u0026minus;\u0026thinsp;3\u0026prime;) (Parada et al., \u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e2016\u003c/span\u003e), following standard instructions. The PCR program consisted of an initial 3-min denaturation at 94\u0026deg;C, followed by 35 cycles with a 45-s denaturation step at 94\u0026deg;C, 1-min hybridization at 55\u0026deg;C, and a 1 min 30 s elongation step at 72\u0026deg;C. Amplification was verified by 1% agarose gel electrophoresis. The PCR1 products were sequenced using Illumina MiSeq 250 \u0026times; 2 bp at the Genotoul platform (Toulouse, France). Amplicon sequence analysis was performed using the QIIME2 pipeline (Bolyen et al., 2019, version 2022.8). Amplicon Sequence Variants (ASVs) were obtained with the DADA2 algorithm; forward and reverse reads were trimmed at 230 and 220 bp, respectively. The expected error rate was set at 2. Reads with a phred score\u0026thinsp;\u0026lt;\u0026thinsp;20 and chimeras were discarded. ASVs were then affiliated taxonomically using the SILVA 138.2\u0026ndash;99 SSU database (Yilmaz et al., \u003cspan citationid=\"CR71\" class=\"CitationRef\"\u003e2014\u003c/span\u003e) and chloroplast- and eukaryote-affiliated reads were discarded. Further analyses were done using R studio and the phyloseq package (McMurdie and Holmes, \u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e2013\u003c/span\u003e). Raw sequences are available at NCBI Sequence Read Archive (Bioproject: PRJNA1266712, samples accession number SAMN56450224 to SAMN56450372).\u003c/p\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003ePhylogeny and genomic organization of symbionts Rubisco encoding genes\u003c/h2\u003e \u003cp\u003eSymbionts\u0026rsquo; Rubisco sequences were extracted from \u003cem\u003eLo. orbiculatus\u003c/em\u003e and \u003cem\u003eLu. borealis\u003c/em\u003e annotated metagenomes (personal data), aligned with 20 relevant amino acids sequences from the literature using ClustalW, and a phylogenetic tree was reconstructed using the maximum likelihood method (JTT model, substitution type: amino acid, rates among site: G; 4. Tree inference options: Nearest-Neighbor-interchange, thread\u0026thinsp;=\u0026thinsp;8). Nodes robustness were evaluated based on 1000 bootstrap replicates using MEGA version 11 (Tamura et al., \u003cspan citationid=\"CR64\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). The 3D structure of Rubisco was predicted based on amino acid sequence using Alphafold (Jumper et al., \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). To visualize possible conformation changes between the different symbionts Rubisco small and large chains, overlapping of both subunits 3D representation has been performed using ChimeraX (Meng et al., \u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e2023\u003c/span\u003e).\u003c/p\u003e \u003c/div\u003e"},{"header":"Results","content":"\u003cdiv id=\"Sec10\" class=\"Section2\"\u003e \u003ch2\u003eIsotopic signatures of Lucinidae during winter\u003c/h2\u003e \u003cp\u003eIsotopic measurements of bivalves collected in 2020 show that \u003cem\u003eTellina sp\u003c/em\u003e. has δ\u0026sup1;\u0026sup3;C isotopic values of -18.7\u0026thinsp;\u0026plusmn;\u0026thinsp;0.3\u0026permil; in the gills and \u0026minus;\u0026thinsp;18.7\u0026thinsp;\u0026plusmn;\u0026thinsp;0.04\u0026permil; in the visceral mass (Vm), respectively. The two Lucinidae species exhibit more negative values. \u003cem\u003eLo. orbiculatus\u003c/em\u003e, displays δ\u0026sup1;\u0026sup3;C values of \u0026minus;\u0026thinsp;31,4\u0026thinsp;\u0026plusmn;\u0026thinsp;1\u0026permil; for the gills and \u0026minus;\u0026thinsp;29.1\u0026thinsp;\u0026plusmn;\u0026thinsp;0.6\u0026permil; for the Vm. \u003cem\u003eLu. borealis\u003c/em\u003e, displays slightly less negative values (-28.5\u0026thinsp;\u0026plusmn;\u0026thinsp;0.7\u0026permil; for the gill and \u0026minus;\u0026thinsp;26.5\u0026thinsp;\u0026plusmn;\u0026thinsp;1.2\u0026permil; in the Vm). In \u003cem\u003eTellina sp\u003c/em\u003e., measured δ\u003csup\u003e15\u003c/sup\u003eN isotopic values are 8.30\u0026thinsp;\u0026plusmn;\u0026thinsp;1.4\u0026permil; for the gills and 8.30\u0026thinsp;\u0026plusmn;\u0026thinsp;1.5\u0026permil; for the Vm. In contrast, both Lucinidae show more negative signatures, \u0026minus;\u0026thinsp;0.71\u0026thinsp;\u0026plusmn;\u0026thinsp;1.7\u0026permil; in the gills and 1.3\u0026thinsp;\u0026plusmn;\u0026thinsp;1.4\u0026permil; in the Vm of \u003cem\u003eLo. orbiculatus\u003c/em\u003e, and \u0026minus;\u0026thinsp;0.03\u0026thinsp;\u0026plusmn;\u0026thinsp;1.5\u0026permil; in the gills and 2.9\u0026thinsp;\u0026plusmn;\u0026thinsp;1.2\u0026permil; in the Vm for \u003cem\u003eLu. borealis\u003c/em\u003e (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA).\u003c/p\u003e \u003cp\u003eFor both Lucinidae species and for both C and N, gill tissues consistently exhibit more depleted isotopic composition than the visceral mass (Wilcoxon rank-sum test, all p\u0026thinsp;\u0026lt;\u0026thinsp;0.0001, Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB-C). Differences were significant for δ\u0026sup1;\u0026sup3;C and δ\u0026sup1;⁵N values between the gill and Vm in both \u003cem\u003eLo. orbiculatus\u003c/em\u003e and \u003cem\u003eLu. borealis\u003c/em\u003e (Wilcoxon rank-sum test, all p\u0026thinsp;\u0026lt;\u0026thinsp;0.0001, Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB-C). The heavy isotope enrichment between visceral mass and gill is around 2\u0026permil; for both nitrogen and carbon. In addition, \u003cem\u003eLucinoma borealis\u003c/em\u003e shows overall less negative isotopic values than \u003cem\u003eLoripes orbiculatus\u003c/em\u003e (Wilcoxon rank-sum test, all p\u0026thinsp;\u0026lt;\u0026thinsp;0.0001; Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB\u0026ndash;C).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003eIsotopic enrichment of lucinid tissues after exposure to labelled microorganisms\u003c/h2\u003e \u003cp\u003eFor both \u003cem\u003eLoripes orbiculatus\u003c/em\u003e and \u003cem\u003eLucinoma borealis\u003c/em\u003e, no significant difference in δ\u0026sup1;\u0026sup3;C or δ\u0026sup1;⁵N values was observed between values measured in T\u003csub\u003e0\u003c/sub\u003e 2020, T\u003csub\u003e0\u003c/sub\u003e 2023 (EC) and experimental controls (LC and LCS), in neither gill nor visceral mass (\u003cem\u003ep\u003c/em\u003e\u003csub\u003e\u003cem\u003eadj\u003c/em\u003e\u003c/sub\u003e \u0026gt; 0.05 for all comparisons; supplementary data S1).\u003c/p\u003e \u003cp\u003eFor \u003cem\u003eLo. orbiculatus\u003c/em\u003e, δ\u0026sup1;\u0026sup3;C values measured in gill tissues of non-exposed controls (EC-g, LC-g, LCS-g) and bacteria-fed individuals (LB-g and LBS-g) were similar (\u003cem\u003ep\u003c/em\u003e\u003csub\u003e\u003cem\u003eadj\u003c/em\u003e\u003c/sub\u003e \u0026gt; 0.05). In contrast, individuals exposed to phytoplankton cultures (LP-g and LPS-g) showed significantly less negative δ\u0026sup1;\u0026sup3;C values (\u003cem\u003ep\u003c/em\u003e\u003csub\u003e\u003cem\u003eadj\u003c/em\u003e\u003c/sub\u003e \u0026lt; 0.05) (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA). In the visceral mass, δ\u0026sup1;\u0026sup3;C values were generally higher than in the gill (\u003cem\u003ep\u003c/em\u003e\u003csub\u003e\u003cem\u003eadj\u003c/em\u003e\u003c/sub\u003e \u0026lt; 0.05). For both tissues, phytoplankton-fed conditions displayed less negative δ\u0026sup1;\u0026sup3;C values than bacteria-fed conditions. With regards to the effect of sulfide, no significant difference in δ\u0026sup1;\u0026sup3;C values was observed between individuals exposed to sulfide and non-sulfide conditions in any given treatment (control, phytoplankton or bacteria) (\u003cem\u003ep\u003c/em\u003e\u003csub\u003e\u003cem\u003eadj\u003c/em\u003e\u003c/sub\u003e \u0026gt; 0.05) (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA).\u003c/p\u003e \u003cp\u003eIn \u003cem\u003eLu. borealis\u003c/em\u003e, no significant differences were observed between controls (EC-g, LC-g, LCS-g) and bacteria-fed conditions (LB-g and LBS-g; \u003cem\u003ep\u003c/em\u003e\u003csub\u003e\u003cem\u003eadj\u003c/em\u003e\u003c/sub\u003e \u0026gt; 0.05). A significant enrichment in \u003csup\u003e13\u003c/sup\u003eC was observed in those exposed to phytoplankton LP-g (δ\u0026sup1;\u0026sup3;C\u0026thinsp;=\u0026thinsp;\u0026minus;\u0026thinsp;22 .32\u0026thinsp;\u0026plusmn;\u0026thinsp;4.64\u0026permil;) and LPS-g (δ\u0026sup1;\u0026sup3;C\u0026thinsp;=\u0026thinsp;\u0026minus;\u0026thinsp;19.69\u0026thinsp;\u0026plusmn;\u0026thinsp;2.96\u0026permil;) (\u003cem\u003ep\u003c/em\u003e\u003csub\u003e\u003cem\u003eadj\u003c/em\u003e\u003c/sub\u003e \u0026lt; 0.05). In the visceral mass, δ\u0026sup1;\u0026sup3;C values increased significantly across all LB-vm, LBS-vm, LP-vm and LPS-vm treatments compared to LC-vm and LCS-vm controls (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eB). These differences were significant in multiple pairwise comparisons (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05 or p\u0026thinsp;\u0026lt;\u0026thinsp;0.01). As for \u003cem\u003eLo. orbiculatus\u003c/em\u003e, the visceral mass showed clearer enrichment in \u003csup\u003e13\u003c/sup\u003eC compared to the gills.\u003c/p\u003e \u003cp\u003eOverall, for both gill and visceral mass, similar trends were thus observed in the two species, with significantly less negative values for \u003cem\u003eLu. borealis\u003c/em\u003e compared to \u003cem\u003eLo. orbiculatus\u003c/em\u003e in all conditions. Overall, highest variations were observed in δ\u0026sup1;\u0026sup3;C values in conditions exposed to phytoplankton, while values did not increase (gill tissue), or only slightly increase (visceral mass) when exposed to bacterial cultures (\u003cem\u003ep\u003c/em\u003e\u003csub\u003e\u003cem\u003eadj\u003c/em\u003e\u003c/sub\u003e \u0026lt; 0.05) (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA-B). In both species and both tissues, addition of sulfide did not lead to significant change in δ\u0026sup1;\u0026sup3;C signatures compared to the sulfide-free condition (LB versus LBS and LP versus LPS).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eIn \u003cem\u003eLo. orbiculatus\u003c/em\u003e gills, δ\u0026sup1;⁵N values in EC-g, LC-g, LCS-g conditions were similar (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA). Conditions exposed to bacteria (LB-g and LBS-g) displayed comparatively higher δ\u0026sup1;⁵N values with LB-g\u0026thinsp;=\u0026thinsp;49.42\u0026thinsp;\u0026plusmn;\u0026thinsp;26.22\u0026permil; and LBS-g\u0026thinsp;=\u0026thinsp;86.19\u0026thinsp;\u0026plusmn;\u0026thinsp;24.81\u0026permil; (\u003cem\u003ep\u003c/em\u003e\u003csub\u003e\u003cem\u003eadj\u003c/em\u003e\u003c/sub\u003e \u0026lt; 0.05). Individuals exposed to phytoplankton (LP-g and LPS-g) exhibited markedly higher δ\u0026sup1;⁵N values compared to all other groups (\u003cem\u003ep\u003c/em\u003e\u003csub\u003e\u003cem\u003eadj\u003c/em\u003e\u003c/sub\u003e \u0026lt; 0.05).\u003c/p\u003e \u003cp\u003eIn the visceral mass, significant increases were observed in all exposed conditions. LB-vm and LBS-vm led to moderate enrichment (\u003cem\u003ep\u003c/em\u003e\u003csub\u003e\u003cem\u003eadj\u003c/em\u003e\u003c/sub\u003e \u0026lt; 0.01). LB-vm δ\u0026sup1;⁵N\u0026thinsp;=\u0026thinsp;84.52\u0026thinsp;\u0026plusmn;\u0026thinsp;80.57\u0026permil; and LBS-vm δ\u0026sup1;⁵N\u0026thinsp;=\u0026thinsp;176.22\u0026thinsp;\u0026plusmn;\u0026thinsp;80.06\u0026permil;. LB-vm and LBS-vm display very important standard deviation indicating high heterogeneity in \u003csup\u003e15\u003c/sup\u003eN incorporation in the visceral mass of \u003cem\u003eLo. orbiculatus\u003c/em\u003e. The highest δ\u0026sup1;⁵N values were measured in phytoplankton-exposed groups LP-vm (δ\u0026sup1;⁵N\u0026thinsp;=\u0026thinsp;734.02\u0026thinsp;\u0026plusmn;\u0026thinsp;213.71\u0026permil;) and LPS-vm (δ\u0026sup1;⁵N\u0026thinsp;=\u0026thinsp;968.03\u0026thinsp;\u0026plusmn;\u0026thinsp;359.42\u0026permil;), both significantly different from all other conditions (\u003cem\u003ep\u003c/em\u003e\u003csub\u003e\u003cem\u003eadj\u003c/em\u003e\u003c/sub\u003e \u0026lt; 0.05), and also displaying high standard deviation (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA).\u003c/p\u003e \u003cp\u003eIn \u003cem\u003eLu. borealis\u003c/em\u003e, δ\u0026sup1;⁵N values in EC-g, LC-g, LCS-g conditions were similar (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eB). Conditions exposed to bacteria and phytoplankton followed the same trend: higher δ\u0026sup1;⁵N values in LB-g (δ\u0026sup1;⁵N\u0026thinsp;=\u0026thinsp;87.14\u0026thinsp;\u0026plusmn;\u0026thinsp;50.93\u0026permil;) and LBS-g (δ\u0026sup1;⁵N\u0026thinsp;=\u0026thinsp;81.82\u0026thinsp;\u0026plusmn;\u0026thinsp;21.99\u0026permil;), and markedly higher values in LP-vm (δ\u0026sup1;⁵N\u0026thinsp;=\u0026thinsp;1040.44\u0026thinsp;\u0026plusmn;\u0026thinsp;484.02\u0026permil;) and LPS-vm (δ\u0026sup1;⁵N\u0026thinsp;=\u0026thinsp;1374.80\u0026thinsp;\u0026plusmn;\u0026thinsp;495.37\u0026permil;) (\u003cem\u003ep\u003c/em\u003e\u003csub\u003e\u003cem\u003eadj\u003c/em\u003e\u003c/sub\u003e \u0026lt; 0.05) compared to EC-g, LC-g, LCS-g conditions (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eB).\u003c/p\u003e \u003cp\u003eIn the visceral mass, specimens exposed to labelled bacterial culture LB-g (δ\u0026sup1;⁵N\u0026thinsp;=\u0026thinsp;173.60\u0026thinsp;\u0026plusmn;\u0026thinsp;53.19\u0026permil;) and LBS-g (δ\u0026sup1;⁵N\u0026thinsp;=\u0026thinsp;192.51\u0026thinsp;\u0026plusmn;\u0026thinsp;90.71\u0026permil;) were significantly enriched compared to controls (\u003cem\u003ep\u003c/em\u003e\u003csub\u003e\u003cem\u003eadj\u003c/em\u003e\u003c/sub\u003e \u0026lt; 0.05). Phytoplankton-fed individuals LP-vm (δ\u0026sup1;⁵N\u0026thinsp;=\u0026thinsp;2027.48\u0026thinsp;\u0026plusmn;\u0026thinsp;419.22\u0026permil;) and LPS-vm (δ\u0026sup1;⁵N\u0026thinsp;=\u0026thinsp;2471.66\u0026thinsp;\u0026plusmn;\u0026thinsp;742.83\u0026permil;) again displayed the highest δ\u0026sup1;⁵N values (\u003cem\u003ep\u003c/em\u003e\u003csub\u003e\u003cem\u003eadj\u003c/em\u003e\u003c/sub\u003e \u0026lt; 0.05), significantly different from all other conditions (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eB).\u003c/p\u003e \u003cp\u003eFor both tissues and lucinid species, no statistical difference was observed between sulfide-free and sulfide condition for both exposed conditions (LB versus LBS and LP versus LPS).\u003c/p\u003e \u003cp\u003eIn summary, across both species, δ\u0026sup1;\u0026sup3;C and δ\u0026sup1;⁵N values were consistently similar among EC-g, LC-g, LCS-g conditions, indicating that the experimental conditions alone did not induce major changes in stable isotope signatures. When comparing bacteria- and phytoplankton-fed conditions, signatures were consistently higher in the latter, evidencing higher enrichment in heavy isotopes when exposed to phytoplankton. Moreover, the addition of sulfide didn\u0026rsquo;t change the isotopic signature in any of the conditions. For δ\u0026sup1;⁵N values, both species and tissues display important standard deviation indicating heterogeneity in \u003csup\u003e15\u003c/sup\u003eN incorporation. The highest δ\u0026sup1;\u0026sup3;C and δ\u0026sup1;⁵N values were always observed in the visceral mass of phytoplankton-fed individuals.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003eRubisco-encoding genes in symbionts metagenomes\u003c/h2\u003e \u003cp\u003eThe phylogenetic tree built based on Rubisco sequences shows that \u003cem\u003erbcL\u003c/em\u003e amino acids sequences from symbionts of both Lucinidae cluster with form I Rubisco of other \u003cem\u003eCa\u003c/em\u003e. Thiodiazotropha, confirming that both symbionts harbor a form I Rubisco (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eA). The annotated genomes of symbionts of \u003cem\u003eLo. orbiculatus\u003c/em\u003e and \u003cem\u003eLu. borealis\u003c/em\u003e both revealed a complete Rubisco operon. The genes encoding the large subunit (\u003cem\u003erbcL\u003c/em\u003e), small subunit (\u003cem\u003erbcS\u003c/em\u003e), and the activation proteins (\u003cem\u003ecbbO\u003c/em\u003e and \u003cem\u003ecbbQ\u003c/em\u003e) were located on the same strand (-), whereas the transcriptional regulator (\u003cem\u003ecbbR\u003c/em\u003e) was located on the opposite strand (+). The genomic arrangement reveals a difference. In \u003cem\u003eLo. orbiculatus\u003c/em\u003e, all genes were present as single copy, whereas in \u003cem\u003eLu. borealis\u003c/em\u003e, the genes encoding activation proteins are present in two copies (\u003cem\u003ecbbO\u003c/em\u003e and \u003cem\u003ecbbQ\u003c/em\u003e, Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eB\u0026ndash;C). Amino acids sequences alignment indicates that the small Rubisco subunit differs by 13.6% between symbionts of \u003cem\u003eLo. orbiculatus\u003c/em\u003e and \u003cem\u003eLu. borealis\u003c/em\u003e, while the large subunits differ by 3.81%. Superimposition of the predicted 3D structures for both the large and small subunits in each symbiont shows high structural similarity, with the main mismatches located at the lateral termini of the proteins (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eD).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003eGill metabolome and associated microbiota\u003c/h2\u003e \u003cp\u003eAnalysis of metabolites composition in gills of both \u003cem\u003eLu. borealis\u003c/em\u003e and \u003cem\u003eLo. orbiculatus\u003c/em\u003e revealed a clear separation between the EC-g (T\u003csub\u003e0\u003c/sub\u003e) group and all other conditions (non-exposed controls LC-g and LCS-g and exposed conditions LB-g; LBS-g; LP-g; LPS-g, Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eA-B). PERMANOVA and pairwise tests confirmed that only the EC-g condition was significatively different from all other conditions. On the other hand, other conditions didn\u0026rsquo;t show significant difference among them.\u003c/p\u003e \u003cp\u003eThe analysis of gill bacterial communities revealed a single dominant ASV in each lucinid, both ASVs matching candidate genus \u003cem\u003eCa.\u003c/em\u003e Thiodiazotropha (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eC). The ASV found in \u003cem\u003eLu. borealis\u003c/em\u003e matched \u003cem\u003eCa\u003c/em\u003e. Thiodiazotropha endolucinoma, previously identified in \u003cem\u003eLucinoma borealis\u003c/em\u003e (GenBank accession number LT548924.1; nucleotide identity\u0026thinsp;=\u0026thinsp;100%), while the ASV found in \u003cem\u003eLo. orbiculatus\u003c/em\u003e matched \u003cem\u003eCa\u003c/em\u003e. Thiodiazotropha endoloripes found in \u003cem\u003eLoripes orbiculatus\u003c/em\u003e (GenBank accession number LT548933.1; nucleotide identity\u0026thinsp;=\u0026thinsp;100%). These two ASVs differ from one another by nine base pairs. After 15 days of experiment, gill bacterial composition remained stable and identical to the EC-g group, and no differences occurred among the different exposure conditions, indicating no sign of bacterial proliferation, bacteriemia, or population switch. Minor ASVs were identified in the condition when Lucinidae were in contact of environmental bacteria (LB-g). For example, three ASVs belonging to genus \u003cem\u003eEndozoicomonas\u003c/em\u003e occurred in low number in \u003cem\u003eLo. orbiculatus\u003c/em\u003e gills. In the LC-g condition, one ASV belonging to the \u003cem\u003eSpirochaeta\u003c/em\u003e genus was found in \u003cem\u003eLo. orbiculatus\u003c/em\u003e gills too.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e"},{"header":"Discussion","content":"\u003cdiv id=\"Sec15\" class=\"Section2\"\u003e \u003ch2\u003eIsotopic signatures support chemoautotrophic nutrition during winter low-sulfide periods\u003c/h2\u003e \u003cp\u003e \u003cem\u003eLoripes orbiculatus\u003c/em\u003e and \u003cem\u003eLucinoma borealis\u003c/em\u003e exhibit δ\u003csup\u003e13\u003c/sup\u003eC and δ\u003csup\u003e15\u003c/sup\u003eN largely more negative than signatures measured in seagrass-beds associated heterotrophic organisms found in Roscoff including \u003cem\u003eTellina\u003c/em\u003e (this study), \u003cem\u003eEnsis ensis\u003c/em\u003e (-18\u0026permil; for δ\u003csup\u003e13\u003c/sup\u003eC and 7.8\u0026permil; for δ\u003csup\u003e15\u003c/sup\u003eN) and \u003cem\u003eLutraria lutraria\u003c/em\u003e (-17,8\u0026permil; for δ\u003csup\u003e13\u003c/sup\u003eC and 9,8\u0026permil; for δ\u003csup\u003e15\u003c/sup\u003eN; Ouisse et al., \u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e2012\u003c/span\u003e). Their δ\u003csup\u003e13\u003c/sup\u003eC values are on the other hand similar to those measured in bivalves harboring autotrophic sulfur-oxidizing symbionts that use a form I RubisCO like \u003cem\u003eSolemya velum\u003c/em\u003e (Say, 1822), δ\u003csup\u003e13\u003c/sup\u003eC = -34 to -30\u0026permil;, and \u003cem\u003eBathymodiolus thermophilus\u003c/em\u003e (Kenk \u0026amp; B. R. Wilson, 1985), δ\u003csup\u003e13\u003c/sup\u003eC = -30\u0026permil;, (Scott et al., \u003cspan citationid=\"CR60\" class=\"CitationRef\"\u003e2004\u003c/span\u003e) as well as other Lucinidea (Duperron et al., \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e2007\u003c/span\u003e). This is congruent with the RubisCO form I operon identified in both lucinid symbionts, that is to date reported in all documented lucinids (Robinson and Cavanaugh, 1995, Duperron et al., \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e2007\u003c/span\u003e; Ratinskaia et al., 2024). This form is composed by 8 large and 8 small subunits and induces an isotopic fractionation about\u0026thinsp;\u0026minus;\u0026thinsp;20\u0026permil; versus source atmospheric CO\u003csub\u003e2\u003c/sub\u003e (δ\u003csup\u003e13\u003c/sup\u003eC\u0026thinsp;~\u0026thinsp;8,5\u0026permil; for dissolved CO\u003csub\u003e2\u003c/sub\u003e in the ocean; O\u0026rsquo;Leary, \u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e1988\u003c/span\u003e), leading to a final signature around ~-30\u0026permil;.\u003c/p\u003e \u003cp\u003eThe visceral mass displays slightly higher δ\u003csup\u003e13\u003c/sup\u003eC values compared to the gill. The ~\u0026thinsp;2\u0026permil; difference is typically found between the gill and the visceral mass in chemosymbiotic bivalves, and interpreted as evidence that carbon is fixed in the gill then transferred to the visceral mass (Hill and McQuaid, \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e2009\u003c/span\u003e; Riou et al., \u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e2010\u003c/span\u003e). These findings confirm that both Lucinidae consume organic compounds produced by their chemosynthetic symbionts, and mainly rely on them even during winter. If sulfide levels are lower in winter, this could lead to slowed down metabolism and reduced carbon turn over in host tissues. Lower carbon turn-over would be consistent with our experimental results in non-fed specimens, that show no significant change in carbon signatures over 15 days.\u003c/p\u003e \u003cp\u003eConsistent differences in δ\u003csup\u003e13\u003c/sup\u003eC and δ\u003csup\u003e15\u003c/sup\u003eN signatures found between \u003cem\u003eLo. orbiculatus\u003c/em\u003e and \u003cem\u003eLu. borealis\u003c/em\u003e (~\u0026thinsp;2\u0026permil; difference) could be due to metabolic differences in host or symbionts physiology, or both. Indeed, \u003cem\u003eLo. orbiculatus\u003c/em\u003e and \u003cem\u003eLu. borealis\u003c/em\u003e have been phylogenetically separated for approximately 100\u0026nbsp;million years (Taylor et al., \u003cspan citationid=\"CR66\" class=\"CitationRef\"\u003e2011\u003c/span\u003e), allowing evolutionary divergence. Besides, and despite that they co-occur, each host harbors a distinct symbiont ASV, indicating high host-symbiont fidelity as well as potential differences. The divergence levels observed in Rubisco sequences could translate into slight functional differences in the CBB cycle, and explain the difference observed in δ\u0026sup1;\u0026sup3;C values, but their highly similar inferred 3D structure does not suggest this. On the other hand, the \u003cem\u003eLo. orbiculatus\u003c/em\u003e symbiont possesses a single copy of \u003cem\u003ecbbO\u003c/em\u003e and \u003cem\u003ecbbQ\u003c/em\u003e, encoding for an AAA+ ATPase and a chaperone protein, respectively, while the \u003cem\u003eLu. borealis\u003c/em\u003e symbiont harbors two copies of both genes. Single copy seems the standard configuration in most autotrophic bacteria (Schwedock et al., \u003cspan citationid=\"CR59\" class=\"CitationRef\"\u003e2004\u003c/span\u003e; Sutter et al., \u003cspan citationid=\"CR63\" class=\"CitationRef\"\u003e2015\u003c/span\u003e). These activase proteins are involved in the post-translational activation of Rubisco by inducing conformational changes and unlocking the active sites (Martinez et al., 2020). The presence of two activase systems could allow different post-translational regulation, and may thus contribute to the differences observed in δ\u0026sup1;\u0026sup3;C values by modulating Rubisco activity and isotopic fractionation in a symbiont-specific manner.\u003c/p\u003e \u003cp\u003eIn plants, the presence of multiple copies of Rubisco activase genes due to events of genome duplication or tandem gene duplication events have been evidenced (Carmo-Silva et al., \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e2015\u003c/span\u003e) and overexpression of Rca lead to crop yield in rice (Fukayama et al., \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e2012\u003c/span\u003e) and may also impact the Rubisco activation in response to temperature (Rundle and Zielinski, \u003cspan citationid=\"CR55\" class=\"CitationRef\"\u003e1991\u003c/span\u003e). In the chemolithoautotroph \u003cem\u003eAcidithiobacillus ferrooxidans\u003c/em\u003e. multiple rubisco-encoding operons have been characterized including two sets of isoforms of CbbQ and CbbO that form hetero-oligomers which act as specific activases for two structurally diverse Rubisco forms (Heinhorst et al., \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e2002\u003c/span\u003e). Similar operons regulated in response to CO\u003csub\u003e2\u003c/sub\u003e concentrations are also present in \u003cem\u003eHydrogenovibrio marinus\u003c/em\u003e (Yoshizawa et al., \u003cspan citationid=\"CR72\" class=\"CitationRef\"\u003e2004\u003c/span\u003e). Complementary analysis on the role and regulation of the Rca and Rubisco in Lucinids are needed\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec16\" class=\"Section2\"\u003e \u003ch2\u003eExperimental evidence for filter-feeding ability\u003c/h2\u003e \u003cp\u003eThe metabolome of both \u003cem\u003eLo. orbiculatus\u003c/em\u003e and \u003cem\u003eLu. borealis\u003c/em\u003e is altered during experiments compared to wild specimens. The metabolomic profile is a proxy for functional status (Watanabe et al., \u003cspan citationid=\"CR70\" class=\"CitationRef\"\u003e2015\u003c/span\u003e; Zhang et al., \u003cspan citationid=\"CR74\" class=\"CitationRef\"\u003e2024\u003c/span\u003e), so this change suggests experimental stress impacting both Lucinidae, which is not unexpected since our lab-based experiments did not replicate real life conditions. However, no mortality was observed during the 15 days of the experiment under any of the conditions. The metabolome composition was on the other hand highly similar among the different experimental conditions, suggesting that the presence of sulfide, bacteria or phytoplankton did not induce further major changes in holobionts functioning. The composition of gill microbiota did not vary either, and in that case remained similar to that of wild specimens, with overwhelming dominance of the species-specific symbiont ASV, and no sign of bacteremia or infection by any particular known pathogen. This suggests that, once acclimated to laboratory conditions, both Lucinidae species were able to maintain a functional status under all conditions, congruent with recent finding that underlined their resistance to starvation (Orgeas-Gobin et al., \u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e2025\u003c/span\u003e). Even in the presence of abundant food sources, symbionts remained the main bacteria in both species.\u003c/p\u003e \u003cp\u003eWhen exposed for 15 days to isotope-labelled phytoplanktonic cells (\u003cem\u003eC. calcitrans\u003c/em\u003e, ~\u0026thinsp;3.5 \u0026micro;m diameter and \u003cem\u003eT. lutea\u003c/em\u003e, ~\u0026thinsp;6 \u0026micro;m), both \u003cem\u003eLucinoma borealis\u003c/em\u003e and \u003cem\u003eLoripes orbiculatus\u003c/em\u003e showed increase in δ\u0026sup1;\u0026sup3;C and δ\u0026sup1;⁵N in both gill and visceral mass. This supports the ability of both species to filter and assimilate phytoplankton-derived organic matter through their digestive system, as in other lamellibranch bivalves. The assimilation of bacteria was less evident, yet δ\u0026sup1;\u0026sup3;C and δ\u0026sup1;⁵N values measured in tissues suggest some assimilation of bacteria-derived material, mainly in the visceral mass as expected for heterotrophy. Overall, results indicate that both lucinid species are able to filter-feed on microorganisms from different sizes classes. Interestingly, the enrichment observed in \u003cem\u003eLu. borealis\u003c/em\u003e was systematically higher than in \u003cem\u003eLo. orbiculatus\u003c/em\u003e. This may suggest interspecific differences in reliance on, or efficiency of, heterotrophic feeding. If confirmed, such differences could imply varying fitness advantages under fluctuating environmental conditions. Altogether, these findings provide experimental evidence for filter-feeding ability in lucinids.\u003c/p\u003e \u003cp\u003eFilter-feeding ability in chemosynthetic bivalves is taxon-dependent. In Vesicomyidae, which inhabit deep-sea environments, the digestive system is highly reduced (Le Pennec and Fiala-M\u0026eacute;dioni, 1988; Le Pennec et al., 1990). \u003cem\u003eBathymodiolus\u003c/em\u003e and \u003cem\u003eIdas\u003c/em\u003e mussels on the other hand possess a reduced yet functional digestive system, with functional labial palps and a stomach containing particles, with secretory and digestive cells indicating a capacity for filtering and digesting external organic matter (Le Pennec et al., 1990, \u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e1995\u003c/span\u003e, Page et al., 2013, Duperron et al., 2010). In the Lucinidae, symbiont-derived nutrition is the main energy source. Yet, their digestive system appears complete and functional (Le Pennec et al., 1990). \u003cem\u003eLoripes orbiculatus\u003c/em\u003e reportedly hosts symbionts within its visceral mass, raising questions about their role in nutrition, infection, and symbiont reacquisition (Alcaraz et al., 2024). Deep-sea Lucinidae, \u003cem\u003eLucinoma aequizonata\u003c/em\u003e and \u003cem\u003eParvilucina tenuisculpta\u003c/em\u003e, were able to ingest radiolabeled particles, demonstrating their potential for filter-feeding (Duplessis et al., \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e2004\u003c/span\u003e). These authors identified a broad range of organisms in the gut of \u003cem\u003eL. aequizonata\u003c/em\u003e, suggesting a non-selective feeding behavior. These findings are in line with our experimental results, which indicate that coastal Lucinidae can consume both bacteria and phytoplankton. Our study shows that coastal Lucinidae are also able to filter-feed. A recent study found that \u003cem\u003eLucinoma capensis\u003c/em\u003e, associated with mangrove sediments, exhibited C and N signatures intermediate between autotrophic and heterotrophic and evidence of diatom ingestion and assimilation, suggesting mixotrophic nutrition which was suggested ot provide dietary supplement to cope with periods of low sulfide availability (Amorim et al., \u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). Our results suggest that this capability also exists in \u003cem\u003eLo. orbiculatus\u003c/em\u003e and \u003cem\u003eLu. borealis\u003c/em\u003e from temperate seagrass beds, but that symbiont-derived nutrition is predominant, \u003cem\u003ein natura\u003c/em\u003e even during low-sulfide periods such as winter. Seagrass-bed sediments are known to harbor high microbial abundances with greater diversity and metabolic activity compared to bare sediments, owing to the presence of marine plants and substantial organic-matter production, which might favor filtration as a primary feeding strategy (Mohapatra et al., \u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e2022\u003c/span\u003e; Zhang et al., \u003cspan citationid=\"CR74\" class=\"CitationRef\"\u003e2024\u003c/span\u003e). However, we propose that free-living microorganisms are relatively scarce and hardly accessible to lucinids in seagrass-bed sediments. Indeed, most microorganisms are either adsorbed onto plant root surfaces, embedded within extracellular polymeric substances, or attached to particles such as microplastics (Battin et al., \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e2016\u003c/span\u003e; Gerbersdorf et al., \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e2020\u003c/span\u003e; Saygin et al., \u003cspan citationid=\"CR56\" class=\"CitationRef\"\u003e2024\u003c/span\u003e), (Zettler et al., \u003cspan citationid=\"CR73\" class=\"CitationRef\"\u003e2013\u003c/span\u003e; Huang et al., \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e2020\u003c/span\u003e; Kreitsberg et al., \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). Therefore, filter-feeding may not represent the optimal strategy for Lucinidae.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec17\" class=\"Section2\"\u003e \u003ch2\u003eMixotrophy in Lucinidae: an adaptive trade-off between energetic efficiency and environmental variability?\u003c/h2\u003e \u003cp\u003eHeterotrophic metabolism is efficient in terms of ATP production but energetically costly due to the complexity of substrate acquisition, digestion, and assimilation (Spietz et al., \u003cspan citationid=\"CR62\" class=\"CitationRef\"\u003e2019\u003c/span\u003e; Nibel et al., 2019; Gralka et al., \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). Despite these costs, heterotrophs often benefit from metabolic networks that can flexibly adjust to changes in substrate, conferring a competitive edge over strictly autotrophic organisms, at least when organic carbon sources are available (Sch\u0026auml;fer-Scherzmann \u0026amp; M\u0026uuml;ller, 2016). In the case of chemosymbiosis, however, the paradigm shifts. Indeed, reliance on symbiont\u0026rsquo;s autotrophy largely alleviates the cost of nutrient acquisition for hosts, as organic carbon is directly produced inside host tissues. We hypothesize that in mixotrophic organisms, autotrophy may offer the host a better energetic yield, because the symbionts carry out the most energy-demanding biochemical reactions. In this scenario, the host can save energy by outsourcing metabolic functions to its symbionts, even if autotrophy is less efficient per unit of chemical substrate (e.g., reduced sulfur compounds) than heterotrophy. However, maintaining the ability to perform facultative filter-feeding may be critical during periods of symbiont loss or low symbiont productivity.\u003c/p\u003e \u003cp\u003eFacultative heterotrophy of animal hosts associated to autotrophic symbionts is observed in reef-building corals. Corals combine autotrophic carbon acquisition from their symbionts with heterotrophic nitrogen uptake via prey capture, highlighting the complementarity of both nutritional strategies (R\u0026auml;decker et al., \u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e2015\u003c/span\u003e). Facultative heterotrophy is also found in bivalves, with some Thyasiridae that host symbionts only facultatively (Batstone et al., \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2014\u003c/span\u003e). In Lucinidae reliance on \u003cem\u003eCa\u003c/em\u003e. Thiodiazotropha, which can fix both carbon and nitrogen (Petersen et al., \u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e2016\u003c/span\u003e; Ratinskaia et al., 2024) should minimize the need for filter-feeding. Maintenance of a functional digestive system in lucinid bivalves despite the establishment of chemosynthetic symbiosis 465\u0026nbsp;million years ago (Taylor et Glover, 2006) suggests that Lucinidae face selective pressure.\u003c/p\u003e \u003cp\u003eWe hypothesize that filter-feeding may be a stress-response mechanism, activated during environmentally challenging periods\u0026mdash;such as low sulfide availability\u0026mdash;or during physiological stress, to support both host and symbiont integrity.\u003c/p\u003e \u003cp\u003eFuture research should aim at identifying the precise environmental conditions and physiological thresholds that trigger a shift to heterotrophic nutrition in lucinid bivalves, in order to further explore the limits of their ecological niche.\u003c/p\u003e \u003c/div\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAcknowledgments\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe thank Lidwine Trouilh (GenoToul platform) for 16S rRNA sequencing and Cedric Leroux (METABOMER mass spectrometry facility) and the metabolomic platform, PtRMN, of the Mus\u0026eacute;um national d\u0026rsquo;Histoire naturelle.\u0026nbsp;We also thank the Roscoff Culture Collection (RCC) and the Roscoff Centre de Ressources Biologiques Marine (CRBM). This study was funded by the Institut de l\u0026rsquo;Oc\u0026eacute;an de l\u0026rsquo;Alliance Sorbonne Universit\u0026eacute;s (SOG grant) and the PPR LIFEDEEPER program (National Research Agency, France 2030: ANR-22-POCE-0007).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData availability statement\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe 16S rRNA gene amplicon datasets generated in this study will be available in the NCBI Sequence Read Archive under BioProject: PRJNA1266712, samples accession number SAMN56450224 to SAMN56450372.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eSupplementary information\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe online version contains supplementary data available at\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConflict of interest\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eGenerative AI statement\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe author(s) declare that no Generative AI was used in the creation of this manuscript.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eAmorim K, Loick-Wilde N, Yuen B, Osvatic JT, W\u0026auml;ge-Recchioni J, Hausmann B, Petersen JM, Fabian J, Wodarg D, Zettler ML (2022) Chemoautotrophy, symbiosis and sedimented diatoms support high biomass of benthic molluscs in the Namibian shelf. Sci Rep 12:9731. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1038/s41598-022-13571-w\u003c/span\u003e\u003cspan address=\"10.1038/s41598-022-13571-w\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eBatstone RT, Laurich JR, Salvo F, Dufour SC (2014) Divergent Chemosymbiosis-Related Characters in Thyasira cf. gouldi (Bivalvia: Thyasiridae). PLoS ONE 9:e92856. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1371/journal.pone.0092856\u003c/span\u003e\u003cspan address=\"10.1371/journal.pone.0092856\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eBattin TJ, Besemer K, Bengtsson MM, Romani AM, Packmann AI (2016) The ecology and biogeochemistry of stream biofilms. Nat Rev Microbiol 14:251\u0026ndash;263. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1038/nrmicro.2016.15\u003c/span\u003e\u003cspan address=\"10.1038/nrmicro.2016.15\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eBolyen E, Rideout JR, Dillon MR, Bokulich NA, Abnet CC, Al-Ghalith GA, Alexander H, Alm EJ, Arumugam M, Asnicar F, Bai Y, Bisanz JE, Bittinger K, Brejnrod A, Brislawn CJ, Brown CT, Callahan BJ, Caraballo-Rodr\u0026iacute;guez AM, Chase J, Cope EK, Da Silva R, Diener C, Dorrestein PC, Douglas GM, Durall DM, Duvallet C, Edwardson CF, Ernst M, Estaki M, Fouquier J, Gauglitz JM, Gibbons SM, Gibson DL, Gonzalez A, Gorlick K, Guo J, Hillmann B, Holmes S, Holste H, Huttenhower C, Huttley GA, Janssen S, Jarmusch AK, Jiang L, Kaehler BD, Kang KB, Keefe CR, Keim P, Kelley ST, Knights D, Koester I, Kosciolek T, Kreps J, Langille MGI, Lee J, Ley R, Liu Y-X, Loftfield E, Lozupone C, Maher M, Marotz C, Martin BD, McDonald D, McIver LJ, Melnik AV, Metcalf JL, Morgan SC, Morton JT, Naimey AT, Navas-Molina JA, Nothias LF, Orchanian SB, Pearson T, Peoples SL, Petras D, Preuss ML, Pruesse E, Rasmussen LB, Rivers A, Robeson MS, Rosenthal P, Segata N, Shaffer M, Shiffer A, Sinha R, Song SJ, Spear JR, Swafford AD, Thompson LR, Torres PJ, Trinh P, Tripathi A, Turnbaugh PJ, Ul-Hasan S, van der Hooft JJJ, Vargas F, V\u0026aacute;zquez-Baeza Y, Vogtmann E, von Hippel M, Walters W, Wan Y, Wang M, Warren J, Weber KC, Williamson AD, Xu ZZ, Zaneveld JR, Zhang Y, Zhu Q., Knight, R., Caporaso, J.G., 2019. Reproducible, interactive, scalable and extensible microbiome data science using QIIME 2. Nat Biotechnol 37, 852\u0026ndash;857. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1038/s41587-019-0209-9\u003c/span\u003e\u003cspan address=\"10.1038/s41587-019-0209-9\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eBreusing C, Xiao Y, Russell SL, Corbett-Detig RB, Li S, Sun J, Chen C, Lan Y, Qian P-Y, Beinart RA (2023) Ecological differences among hydrothermal vent symbioses may drive contrasting patterns of symbiont population differentiation. mSystems 8:e0028423. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1128/msystems.00284-23\u003c/span\u003e\u003cspan address=\"10.1128/msystems.00284-23\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eBrodersen KE, Hammer KJ, Schrameyer V, Floytrup A, Rasheed MA, Ralph PJ, K\u0026uuml;hl M, Pedersen O (2017) Sediment Resuspension and Deposition on Seagrass Leaves Impedes Internal Plant Aeration and Promotes Phytotoxic H2S Intrusion. Front Plant Sci 8:657. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.3389/fpls.2017.00657\u003c/span\u003e\u003cspan address=\"10.3389/fpls.2017.00657\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eCarmo-Silva E, Scales JC, Madgwick PJ, Parry MAJ (2015) Optimizing Rubisco and its regulation for greater resource use efficiency. Plant Cell Environ 38:1817\u0026ndash;1832. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1111/pce.12425\u003c/span\u003e\u003cspan address=\"10.1111/pce.12425\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eCaro A, Got P, Bouvy M, Troussellier M, Gros O (2009) Effects of long-term starvation on a host bivalve (Codakia orbicularis, Lucinidae) and its symbiont population. Appl Environ Microbiol 75:3304\u0026ndash;3313. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1128/AEM.02659-08\u003c/span\u003e\u003cspan address=\"10.1128/AEM.02659-08\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eCheng L, Normandeau C, Bowden R, Doucett R, Gallagher B, Gillikin DP, Kumamoto Y, McKay JL, Middlestead P, Ninnemann U, Nothaft D, Dubinina EO, Quay P, Reverdin G, Shirai K, M\u0026oslash;rkved PT, Theiling BP, van Geldern R, Wallace DWR (2019) An international intercomparison of stable carbon isotope composition measurements of dissolved inorganic carbon in seawater. Limnol Oceanography: Methods 17:200\u0026ndash;209. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1002/lom3.10300\u003c/span\u003e\u003cspan address=\"10.1002/lom3.10300\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eConway N, Capuzzo JM, Fry B (1989) The role of endosymbiotic bacteria in the nutrition of Solemya velum: Evidence from a stable isotope analysis of endosymbionts and host. Limnol Oceanogr 34:249\u0026ndash;255. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.4319/lo.1989.34.1.0249\u003c/span\u003e\u003cspan address=\"10.4319/lo.1989.34.1.0249\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eCotovicz LC, Knoppers BA, Deirmendjian L, Abril G (2019) Sources and sinks of dissolved inorganic carbon in an urban tropical coastal bay revealed by δ13C-DIC signals. Estuarine. Coastal Shelf Sci 220:185\u0026ndash;195. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1016/j.ecss.2019.02.048\u003c/span\u003e\u003cspan address=\"10.1016/j.ecss.2019.02.048\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eDescolas-Gros C, Fontungne M (1990) Stable carbon isotope fractionation by marine phytoplankton during photosynthesis. Plant Cell Environ 13:207\u0026ndash;218. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1111/j.1365-3040.1990.tb01305.x\u003c/span\u003e\u003cspan address=\"10.1111/j.1365-3040.1990.tb01305.x\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eDubilier N, Bergin C, Lott C (2008) Symbiotic diversity in marine animals: the art of harnessing chemosynthesis. Nat Rev Microbiol 6:725\u0026ndash;740. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1038/nrmicro1992\u003c/span\u003e\u003cspan address=\"10.1038/nrmicro1992\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eDufour SC (2005) Gill anatomy and the evolution of symbiosis in the bivalve family Thyasiridae. Biol Bull 208:200\u0026ndash;212. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.2307/3593152\u003c/span\u003e\u003cspan address=\"10.2307/3593152\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eDuperron S, Fiala-M\u0026eacute;dioni A, Caprais J-C, Olu K, Sibuet M (2007) Evidence for chemoautotrophic symbiosis in a Mediterranean cold seep clam (Bivalvia: Lucinidae): comparative sequence analysis of bacterial 16S rRNA, APS reductase and RubisCO genes. FEMS Microbiol Ecol 59:64\u0026ndash;70. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1111/j.1574-6941.2006.00194.x\u003c/span\u003e\u003cspan address=\"10.1111/j.1574-6941.2006.00194.x\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eDuperron S, Foucault P, Duval C, Goto M, Gallet A, Colas S, Marie B (2023) Multi-omics analyses from a single sample: prior metabolite extraction does not alter the 16S rRNA-based characterization of prokaryotic community in a diversity of sample types. FEMS Microbiol Lett 370:fnad125. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1093/femsle/fnad125\u003c/span\u003e\u003cspan address=\"10.1093/femsle/fnad125\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eDuplessis MR, Dufour SC, Blankenship LE, Felbeck H, Yayanos AA (2004) Anatomical and experimental evidence for particulate feeding in Lucinoma aequizonataand Parvilucina tenuisculpta (Bivalvia: Lucinidae) from the Santa Barbara Basin. Mar Biol 145:551\u0026ndash;561. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1007/s00227-004-1350-6\u003c/span\u003e\u003cspan address=\"10.1007/s00227-004-1350-6\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eElisabeth NH, Caro A, C\u0026eacute;saire T, Mansot J-L, Escalas A, Sylvestre M-N, Jean-Louis P, Gros O (2014) Comparative modifications in bacterial gill-endosymbiotic populations of the two bivalves Codakia orbiculata and Lucina pensylvanica during bacterial loss and reacquisition. FEMS Microbiol Ecol 89:646\u0026ndash;658. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1111/1574-6941.12366\u003c/span\u003e\u003cspan address=\"10.1111/1574-6941.12366\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eFeng D, Peckmann J, Li N, Kiel S, Qiu J-W, Liang Q, Carney RS, Peng Y, Tao J, Chen D (2018) The stable isotope fingerprint of chemosymbiosis in the shell organic matrix of seep-dwelling bivalves. Chem Geol 479:241\u0026ndash;250. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1016/j.chemgeo.2018.01.015\u003c/span\u003e\u003cspan address=\"10.1016/j.chemgeo.2018.01.015\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eFukayama H, Ueguchi C, Nishikawa K, Katoh N, Ishikawa C, Masumoto C, Hatanaka T, Misoo S (2012) Overexpression of rubisco activase decreases the photosynthetic CO2 assimilation rate by reducing rubisco content in rice leaves. Plant Cell Physiol 53:976\u0026ndash;986. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1093/pcp/pcs042\u003c/span\u003e\u003cspan address=\"10.1093/pcp/pcs042\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eGerbersdorf SU, Koca K, de Beer D, Chennu A, Noss C, Risse-Buhl U, Weitere M, Eiff O, Wagner M, Aberle J, Schweikert M, Terheiden K (2020) Exploring flow-biofilm-sediment interactions: Assessment of current status and future challenges. Water Res 185:116182. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1016/j.watres.2020.116182\u003c/span\u003e\u003cspan address=\"10.1016/j.watres.2020.116182\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eGralka M, Pollak S, Cordero OX (2023) Genome content predicts the carbon catabolic preferences of heterotrophic bacteria. Nat Microbiol 8:1799\u0026ndash;1808. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1038/s41564-023-01458-z\u003c/span\u003e\u003cspan address=\"10.1038/s41564-023-01458-z\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eHeinhorst S, Baker SH, Johnson DR, Davies PS, Cannon GC, Shively JM (2002) Two Copies of form I RuBisCO genes in Acidithiobacillus ferrooxidans ATCC 23270. Curr Microbiol 45:115\u0026ndash;117. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1007/s00284-001-0094-5\u003c/span\u003e\u003cspan address=\"10.1007/s00284-001-0094-5\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eHill JM, McQuaid CD (2009) Effects of food quality on tissue-specific isotope ratios in the mussel Perna perna. Hydrobiologia 635:81\u0026ndash;94. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1007/s10750-009-9865-y\u003c/span\u003e\u003cspan address=\"10.1007/s10750-009-9865-y\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eHuang Y, Xiao X, Xu C, Perianen YD, Hu J, Holmer M (2020) Seagrass beds acting as a trap of microplastics - Emerging hotspot in the coastal region? Environ Pollut 257:113450. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1016/j.envpol.2019.113450\u003c/span\u003e\u003cspan address=\"10.1016/j.envpol.2019.113450\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eJumper J, Evans R, Pritzel A, Green T, Figurnov M, Ronneberger O, Tunyasuvunakool K, Bates R, Ž\u0026iacute;dek A, Potapenko A, Bridgland A, Meyer C, Kohl SAA, Ballard AJ, Cowie A, Romera-Paredes B, Nikolov S, Jain R, Adler J, Back T, Petersen S, Reiman D, Clancy E, Zielinski M, Steinegger M, Pacholska M, Berghammer T, Bodenstein S, Silver D, Vinyals O, Senior AW, Kavukcuoglu K, Kohli P, Hassabis D (2021) Highly accurate protein structure prediction with AlphaFold. Nature 596:583\u0026ndash;589. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1038/s41586-021-03819-2\u003c/span\u003e\u003cspan address=\"10.1038/s41586-021-03819-2\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eKenk VC, Wilson B (1985) A new mussel (Bivalvia, Mytilidae) from hydrothermal vents in the Galapagos Rift zone. Malacologia\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eKoch M, Johnson C, Madden C, Pedersen O (2022) Irradiance, Water Column O2, and Tide Drive Internal O2 Dynamics and Meristem H2S Detection in the Dominant Caribbean-Tropical Atlantic Seagrass, Thalassia testudinum. Estuaries Coasts 45. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1007/s12237-022-01064-y\u003c/span\u003e\u003cspan address=\"10.1007/s12237-022-01064-y\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eKreitsberg R, Raudna-Kristoffersen M, Heinlaan M, Ward R, Visnapuu M, Kisand V, Meitern R, Kotta J, Tuvikene A (2021) Seagrass beds reveal high abundance of microplastic in sediments: A case study in the Baltic Sea. Mar Pollut Bull 168:112417. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1016/j.marpolbul.2021.112417\u003c/span\u003e\u003cspan address=\"10.1016/j.marpolbul.2021.112417\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eKrueger T, Bodin J, Horwitz N, Loussert-Fonta C, Sakr A, Escrig S, Fine M, Meibom A (2018) Temperature and feeding induce tissue level changes in autotrophic and heterotrophic nutrient allocation in the coral symbiosis - A NanoSIMS study. Sci Rep 8:12710. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1038/s41598-018-31094-1\u003c/span\u003e\u003cspan address=\"10.1038/s41598-018-31094-1\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLe Pennec M, Beninger PG, Herry A (1995) Feeding and digestive adaptations of bivalve molluscs to sulphide-rich habitats. Comp Biochem Physiol Part A: Physiol 111:183\u0026ndash;189. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1016/0300-9629(94)00211-B\u003c/span\u003e\u003cspan address=\"10.1016/0300-9629(94)00211-B\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLe Pennec M, Donval A, Herry A (1990a) Nutritional strategies of the hydrothermal ecosystem bivalves. Progress in Oceanography. Deep-Sea Biology 24:71\u0026ndash;80. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1016/0079-6611(90)90020-3\u003c/span\u003e\u003cspan address=\"10.1016/0079-6611(90)90020-3\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLepennec M, Fialamedioni A (1988) The role of the digestive-tract of calyptogena-laubieri and calyptogena-phaseoliformis, vesicomyid bivalves of the subduction zones of japan. Oceanol Acta 11:193\u0026ndash;199\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLeroy F, Riera P, Jeanthon C, Edmond F, Leroux C, Comtet T (2012) Importance of bacterivory and preferential selection toward diatoms in larvae of \u003cem\u003eCrepidula fornicata\u003c/em\u003e (L.) assessed by a dual stable isotope (13C, 15N) labeling approach. J Sea Res 70:23\u0026ndash;31. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1016/j.seares.2012.02.006\u003c/span\u003e\u003cspan address=\"10.1016/j.seares.2012.02.006\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003evon Linn\u0026eacute; C, von Linn\u0026eacute; C, Salvius L (1758) Caroli Linnaei\u0026hellip; Systema naturae per regna tria naturae:secundum classes, ordines, genera, species, cum characteribus, differentiis, synonymis, locis. Impensis Direct. Laurentii Salvii, Holmiae. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.5962/bhl.title.542\u003c/span\u003e\u003cspan address=\"10.5962/bhl.title.542\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMartinez S, Grover R, Ferrier-Pag\u0026egrave;s C (2024) Unveiling the importance of heterotrophy for coral symbiosis under heat stress. mBio 15:e0196624. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1128/mbio.01966-24\u003c/span\u003e\u003cspan address=\"10.1128/mbio.01966-24\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMcMurdie PJ, Holmes S (2013) phyloseq: an R package for reproducible interactive analysis and graphics of microbiome census data. PLoS ONE 8:e61217. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1371/journal.pone.0061217\u003c/span\u003e\u003cspan address=\"10.1371/journal.pone.0061217\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMeng EC, Goddard TD, Pettersen EF, Couch GS, Pearson ZJ, Morris JH, Ferrin TE (2023) UCSF ChimeraX: Tools for structure building and analysis. Protein Sci 32:e4792. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1002/pro.4792\u003c/span\u003e\u003cspan address=\"10.1002/pro.4792\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMohapatra M, Manu S, Dash SP, Rastogi G (2022) Seagrasses and local environment control the bacterial community structure and carbon substrate utilization in brackish sediments. J Environ Manage 314:115013. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1016/j.jenvman.2022.115013\u003c/span\u003e\u003cspan address=\"10.1016/j.jenvman.2022.115013\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eNeufeld Z, Haynes P, Garcon V, Sudre J (2002) Ocean fertilization experiments may initiate a large scale phytoplankton bloom. Geophys Res Lett - GEOPHYS RES LETT 29. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1029/2001GL013677\u003c/span\u003e\u003cspan address=\"10.1029/2001GL013677\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eNiebel B, Leupold S, Heinemann M (2019) An upper limit on Gibbs energy dissipation governs cellular metabolism. Nat Metab 1:125\u0026ndash;132. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1038/s42255-018-0006-7\u003c/span\u003e\u003cspan address=\"10.1038/s42255-018-0006-7\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eO\u0026rsquo;Leary MH (1988) Carbon Isotopes in Photosynthesis: Fractionation techniques may reveal new aspects of carbon dynamics in plants. Bioscience 38:328\u0026ndash;336. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.2307/1310735\u003c/span\u003e\u003cspan address=\"10.2307/1310735\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eOrgeas-Gobin S, Piquet B, Marie B, Andersen AC, Tanguy A, Duperron S (2025) Symbiont retention and holobiont response under simulated sulfide deprivation in Lucinid clams from seagrass beds. Front Microbiol 16:1637201. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.3389/fmicb.2025.1637201\u003c/span\u003e\u003cspan address=\"10.3389/fmicb.2025.1637201\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003ePMID: 41472809; PMCID: PMC12746666\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eOsvatic JT, Yuen B, Kunert M, Wilkins L, Hausmann B, Girguis P, Lundin K, Taylor J, Jospin G, Petersen JM (2023) Gene loss and symbiont switching during adaptation to the deep sea in a globally distributed symbiosis. ISME J 17:453\u0026ndash;466. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1038/s41396-022-01355-z\u003c/span\u003e\u003cspan address=\"10.1038/s41396-022-01355-z\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eOuisse V, Riera P, Mign\u0026eacute; A, Leroux C, Davoult D (2012) Food web analysis in intertidal Zostera marina and Zostera noltii communities in winter and summer. Mar Biol 159:165\u0026ndash;175. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1007/s00227-011-1796-2\u003c/span\u003e\u003cspan address=\"10.1007/s00227-011-1796-2\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003ePage HM, Fisher CR, Childress JJ (1990) Role of filter-feeding in the nutritional biology of a deep-sea mussel with methanotrophic symbionts. Mar Biol 104:251\u0026ndash;257. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1007/BF01313266\u003c/span\u003e\u003cspan address=\"10.1007/BF01313266\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003ePales Espinosa E, Tanguy A, Le Panse S, Lallier F, Allam B, Boutet I (2013) Endosymbiotic bacteria in the bivalve \u003cem\u003eLoripes lacteus\u003c/em\u003e: Localization, characterization and aspects of symbiont regulation. J Exp Mar Biol Ecol 448:327\u0026ndash;336. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1016/j.jembe.2013.07.015\u003c/span\u003e\u003cspan address=\"10.1016/j.jembe.2013.07.015\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eParada AE, Needham DM, Fuhrman JA (2016) Every base matters: assessing small subunit rRNA primers for marine microbiomes with mock communities, time series and global field samples. Environ Microbiol 18:1403\u0026ndash;1414. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1111/1462-2920.13023\u003c/span\u003e\u003cspan address=\"10.1111/1462-2920.13023\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003ePetersen JM, Kemper A, Gruber-Vodicka H, Cardini U, van der Geest M, Kleiner M, Bulgheresi S, Mu\u0026szlig;mann M, Herbold C, Seah BKB, Antony CP, Liu D, Belitz A, Weber M (2016) Chemosynthetic symbionts of marine invertebrate animals are capable of nitrogen fixation. Nat Microbiol 2:16195. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1038/nmicrobiol.2016.195\u003c/span\u003e\u003cspan address=\"10.1038/nmicrobiol.2016.195\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003ePetersen JM, Zielinski FU, Pape T, Seifert R, Moraru C, Amann R, Hourdez S, Girguis PR, Wankel SD, Barbe V, Pelletier E, Fink D, Borowski C, Bach W, Dubilier N (2011) Hydrogen is an energy source for hydrothermal vent symbioses. Nature 476:176\u0026ndash;180. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1038/nature10325\u003c/span\u003e\u003cspan address=\"10.1038/nature10325\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eR\u0026auml;decker N, Pogoreutz C, Voolstra CR, Wiedenmann J, Wild C (2015) Nitrogen cycling in corals: the key to understanding holobiont functioning? Trends Microbiol 23:490\u0026ndash;497. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1016/j.tim.2015.03.008\u003c/span\u003e\u003cspan address=\"10.1016/j.tim.2015.03.008\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eRatinskaia L, Malavin S, Zvi-Kedem T, Vintila S, Kleiner M, Rubin-Blum M (2024a) Metabolically-versatile Ca. Thiodiazotropha symbionts of the deep-sea lucinid clam Lucinoma kazani have the genetic potential to fix nitrogen. ISME Commun 4:ycae076. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1093/ismeco/ycae076\u003c/span\u003e\u003cspan address=\"10.1093/ismeco/ycae076\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eRiou V, Duperron S, Halary S, Dehairs F, Bouillon S, Martins I, Cola\u0026ccedil;o A, Serr\u0026atilde;o Santos R (2010) Variation in physiological indicators in \u003cem\u003eBathymodiolus azoricus\u003c/em\u003e (Bivalvia: Mytilidae) at the Menez Gwen Mid-Atlantic Ridge deep-sea hydrothermal vent site within a year. Mar Environ Res 70:264\u0026ndash;271. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1016/j.marenvres.2010.05.008\u003c/span\u003e\u003cspan address=\"10.1016/j.marenvres.2010.05.008\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eRobinson JJ, Scott KM, Swanson ST, O\u0026rsquo;Leary MH, Horken K, Tabita FR, Cavanaugh CM (2003) Kinetic isotope effect and characterization of form II RubisCO from the chemoautotrophic endosymbionts of the hydrothermal vent tubeworm Riftia pachyptila. Limnol Oceanogr 48:48\u0026ndash;54. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.4319/lo.2003.48.1.0048\u003c/span\u003e\u003cspan address=\"10.4319/lo.2003.48.1.0048\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eRundle SJ, Zielinski RE (1991) Organization and expression of two tandemly oriented genes encoding ribulosebisphosphate carboxylase/oxygenase activase in barley. J Biol Chem 266:4677\u0026ndash;4685\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSaygin H, Tilkili B, Kayisoglu P, Baysal A (2024) Oxidative stress, biofilm-formation and activity responses of \u003cem\u003eP. aeruginosa\u003c/em\u003e to microplastic-treated sediments: Effect of temperature and sediment type. Environ Res 248:118349. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1016/j.envres.2024.118349\u003c/span\u003e\u003cspan address=\"10.1016/j.envres.2024.118349\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSchrameyer V, York PH, Chartrand K, Ralph PJ, K\u0026uuml;hl M, Brodersen KE, Rasheed MA (2018) Contrasting impacts of light reduction on sediment biogeochemistry in deep- and shallow-water tropical seagrass assemblages (Green Island, Great Barrier Reef). Mar Environ Res 136:38\u0026ndash;47. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1016/j.marenvres.2018.02.008\u003c/span\u003e\u003cspan address=\"10.1016/j.marenvres.2018.02.008\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSchuchmann K, M\u0026uuml;ller V (2016) Energetics and Application of Heterotrophy in Acetogenic Bacteria. Appl Environ Microbiol 82:4056\u0026ndash;4069. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1128/AEM.00882-16\u003c/span\u003e\u003cspan address=\"10.1128/AEM.00882-16\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSchwedock J, Harmer TL, Scott KM, Hektor HJ, Seitz AP, Fontana MC, Distel DL, Cavanaugh CM (2004) Characterization and expression of genes from the RubisCO gene cluster of the chemoautotrophic symbiont of Solemya velum: cbbLSQO. Arch Microbiol 182:18\u0026ndash;29. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1007/s00203-004-0689-x\u003c/span\u003e\u003cspan address=\"10.1007/s00203-004-0689-x\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eScott KM, Schwedock J, Schrag DP, Cavanaugh CM (2004) Influence of form IA RubisCO and environmental dissolved inorganic carbon on the delta13C of the clam-chemoautotroph symbiosis Solemya velum. Environ Microbiol 6:1210\u0026ndash;1219. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1111/j.1462-2920.2004.00642.x\u003c/span\u003e\u003cspan address=\"10.1111/j.1462-2920.2004.00642.x\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSogin EM, Leisch N, Dubilier N (2020) Chemosynthetic symbioses. Curr Biol 30:R1137\u0026ndash;R1142. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1016/j.cub.2020.07.050\u003c/span\u003e\u003cspan address=\"10.1016/j.cub.2020.07.050\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSpietz RL, Lundeen RA, Zhao X, Nicastro D, Ingalls AE, Morris RM (2019) Heterotrophic carbon metabolism and energy acquisition in Candidatus Thioglobus singularis strain PS1, a member of the SUP05 clade of marine Gammaproteobacteria. Environ Microbiol 21:2391\u0026ndash;2401. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1111/1462-2920.14623\u003c/span\u003e\u003cspan address=\"10.1111/1462-2920.14623\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSutter M, Roberts EW, Gonzalez RC, Bates C, Dawoud S, Landry K, Cannon GC, Heinhorst S, Kerfeld CA (2015) Structural Characterization of a Newly Identified Component of α-Carboxysomes: The AAA+ Domain Protein CsoCbbQ. Sci Rep 5:16243. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1038/srep16243\u003c/span\u003e\u003cspan address=\"10.1038/srep16243\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eTamura K, Stecher G, Kumar S (2021) Mol Biol Evol 38:3022\u0026ndash;3027. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1093/molbev/msab120\u003c/span\u003e\u003cspan address=\"10.1093/molbev/msab120\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e. MEGA11: Molecular Evolutionary Genetics Analysis Version 11\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eTaylor JD, Glover EA (2000) Functional anatomy, chemosymbiosis and evolution of the Lucinidae. Geological Society, London, Special Publications 177, 207\u0026ndash;225. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1144/GSL.SP.2000.177.01.12\u003c/span\u003e\u003cspan address=\"10.1144/GSL.SP.2000.177.01.12\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eTaylor JD, Glover EA, Smith L, Dyal P, Williams ST (2011) Molecular phylogeny and classification of the chemosymbiotic bivalve family Lucinidae (Mollusca: Bivalvia). Zool J Linn Soc 163:15\u0026ndash;49. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1111/j.1096-3642.2011.00700.x\u003c/span\u003e\u003cspan address=\"10.1111/j.1096-3642.2011.00700.x\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eThorsen S, Kristensen E, Valdemarsen T, Flindt M, Organo Quintana C, Holmer M (2019) Fertilizer-derived N in opportunistic macroalgae after flooding of agricultural land. Mar Ecol Prog Ser 616. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.3354/meps12927\u003c/span\u003e\u003cspan address=\"10.3354/meps12927\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eThubaut J, Corbari L, Gros O, Duperron S, Couloux A, Samadi S (2013a) Integrative biology of Idas iwaotakii (Habe, 1958), a model species associated with sunken organic substrates. PLoS ONE 8:e69680. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1371/journal.pone.0069680\u003c/span\u003e\u003cspan address=\"10.1371/journal.pone.0069680\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eTsai Y-CC, Ye F, Liew L, Liu D, Bhushan S, Gao Y-G, Mueller-Cajar O (2020) Insights into the mechanism and regulation of the CbbQO-type Rubisco activase, a MoxR AAA+ ATPase. Proc Natl Acad Sci U S A 117:381\u0026ndash;387. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1073/pnas.1911123117\u003c/span\u003e\u003cspan address=\"10.1073/pnas.1911123117\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eWatanabe M, Meyer KA, Jackson TM, Schock TB, Johnson WE, Bearden DW (2015) Application of NMR-based metabolomics for environmental assessment in the Great Lakes using zebra mussel (Dreissena polymorpha). Metabolomics 11:1302\u0026ndash;1315. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1007/s11306-015-0789-4\u003c/span\u003e\u003cspan address=\"10.1007/s11306-015-0789-4\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eYilmaz P, Parfrey LW, Yarza P, Gerken J, Pruesse E, Quast C, Schweer T, Peplies J, Ludwig W, Gl\u0026ouml;ckner FO (2014) The SILVA and All-species Living Tree Project (LTP) taxonomic frameworks. Nucleic Acids Res 42:D643\u0026ndash;648. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1093/nar/gkt1209\u003c/span\u003e\u003cspan address=\"10.1093/nar/gkt1209\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eYoshizawa Y, Toyoda K, Arai H, Ishii M, Igarashi Y (2004) CO2-responsive expression and gene organization of three ribulose-1,5-bisphosphate carboxylase/oxygenase enzymes and carboxysomes in Hydrogenovibrio marinus strain MH-110. J Bacteriol 186:5685\u0026ndash;5691. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1128/JB.186.17.5685-5691.2004\u003c/span\u003e\u003cspan address=\"10.1128/JB.186.17.5685-5691.2004\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eZettler ER, Mincer TJ, Amaral-Zettler LA (2013) Life in the plastisphere: microbial communities on plastic marine debris. Environ Sci Technol 47:7137\u0026ndash;7146. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1021/es401288x\u003c/span\u003e\u003cspan address=\"10.1021/es401288x\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eZhang Y, Wang H, Liu S, Kong X, Chang L, Zhao L, Bao Z, Hu X (2024) Multi-tissue metabolomic profiling reveals the crucial metabolites and pathways associated with scallop growth. BMC Genomics 25:1091. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1186/s12864-024-11016-4\u003c/span\u003e\u003cspan address=\"10.1186/s12864-024-11016-4\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eZhang Y, Wang Q, Yao Y, Tan F, Jiang L, Shi W, Yang W, Liu J (2024) Bacterial Communities in Zostera marina Seagrass Beds of Northern China. Water 16:935. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.3390/w16070935\u003c/span\u003e\u003cspan address=\"10.3390/w16070935\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"marine-biology","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"mabi","sideBox":"Learn more about [Marine Biology](https://www.springer.com/journal/227)","snPcode":"227","submissionUrl":"https://submission.nature.com/new-submission/227/3","title":"Marine Biology","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"Isotopy, Mixotrophy, Seagrass bed, Lucinoma borealis, Loripes orbiculatus","lastPublishedDoi":"10.21203/rs.3.rs-9360400/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-9360400/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eBivalves of the family Lucinidae, \u003cem\u003eLoripes orbiculatus\u003c/em\u003e and \u003cem\u003eLucinoma borealis\u003c/em\u003e, are sympatric species inhabiting coastal seagrass beds in Roscoff Bay. These bivalves harbor chemoautotrophic symbionts within their gills that provide autotrophic nutrition to the host by oxidizing hydrogen sulfide (H₂S) present in the sediment. Although Lucinidae are typically considered fully autotrophic in these environments, seagrass beds are subject to fluctuations in sulfide availability due to tides, seasonal changes, and anthropogenic disturbances. This study investigates how Lucinidae cope with periods of low sulfide availability by exploring their nutritional strategies under sulfide starvation. Lucinidae species were incubated for 15 days in the presence of sediment bacteria or a mixture of two phytoplankton species labeled with \u0026sup1;⁵N and \u0026sup1;\u0026sup3;C, with or without addition of sulfide, to trace assimilation pathways into the gill and visceral mass. Results show that both \u0026sup1;⁵N and \u0026sup1;\u0026sup3;C were incorporated into tissues within seven days, indicating that lucinids are capable of assimilating both autotrophy- and heterotrophy-derived sources of nutrition. Composition of their associated bacterial communities was not affected. These findings provide evidence of mixotrophy in coastal Lucinidae, indicating that they can shift to filter-feeding under low sulfide availability, probably contributing to their ecological success. Nutritional plasticity of the Lucinidae may be key to their resilience in fluctuating coastal environments.\u003c/p\u003e","manuscriptTitle":"Experimental evidence of mixotrophy in seagrass-associated Lucinidae in the absence of sulfide","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2026-05-06 10:48:34","doi":"10.21203/rs.3.rs-9360400/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"reviewerAgreed","content":"","date":"2026-04-28T05:06:28+00:00","index":0,"fulltext":""},{"type":"reviewersInvited","content":"","date":"2026-04-28T03:23:20+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2026-04-20T05:20:45+00:00","index":"","fulltext":""},{"type":"submitted","content":"Marine Biology","date":"2026-04-08T14:48:01+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"marine-biology","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"mabi","sideBox":"Learn more about [Marine Biology](https://www.springer.com/journal/227)","snPcode":"227","submissionUrl":"https://submission.nature.com/new-submission/227/3","title":"Marine Biology","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"35eeea1e-de5a-4f80-b75c-e4a4e83fe6e4","owner":[],"postedDate":"May 6th, 2026","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"under-review","subjectAreas":[],"tags":[],"updatedAt":"2026-05-06T10:48:34+00:00","versionOfRecord":[],"versionCreatedAt":"2026-05-06 10:48:34","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-9360400","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-9360400","identity":"rs-9360400","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}