SUPPORTING INFORMATION
Distance interaction between marine cave-dwelling sponges and crustaceans
Mathieu Santonja1,2, Stéphane Greff1, Marie Le Croller1, Olivier P. Thomas1,3, Thierry Pérez1*
1. Institut Méditerranéen de Biodiversité et d’Ecologie Marine et Continentale (IMBE), UMR 7263 CNRS, IRD, Aix Marseille Université, Avignon Université, Station Marine d’Endoume, rue de la Batterie des Lions, 13007 Marseille, France. 2. Univ Rennes, CNRS, ECOBIO - UMR 6553, F-35000 Rennes, France. 3. Marine Biodiscovery Laboratory, School of Chemistry and Ryan Institute, National University of Ireland, Galway (NUI Galway), University Road, H91 TK33 Galway, Ireland.
*Corresponding author:
[email protected]
Supplementary Fig. S1. The four selected sponge species representatives of the semi-dark benthic community in underwater caves: (a) Aplysina cavernicola forming tubes up to 15 cm high and covering a surface of up to 30 cm in diameter, (b) Haliclona fulva forming crusts up to 1-2 cm thick and covering a surface of up to 15 cm in diameter, (c) Oscarella tuberculata, thin and lobate, covering a surface of up to 20 cm in diameter, and (d) Spongia officinalis, usually massive and globulous, measuring up to 30 cm in diameter.
Supplementary Fig. S2. The four crustaceans, with contrasting ecological habits, selected to perform the chemosensory trials: (a) Hemimysis margalefi, (b) Leptomysis sp., (c) Palaemon elegans and (d) Palaemon serratus. Scale bar = 2 mm.
Supplementary Fig. S3. Two-channel choice experimental device used to measure crustacean behavioural responses facing seawater samples containing (a) sponge chemical cues, here visualized by fluorescein, or (b) control seawater without sponge chemical cues. (c) represents the downstream compartment and (d) is the opening hole through which crustaceans were introduced.
Supplementary Fig. S4. Response of the four crustaceans from seawater control in both upstream channels (i.e. in absence of manipulated cues). Results are mean time (± SE) in second that each species spent in the left (black bar) and the right (white bar) channels. HM = Hemimysis margalefi, LS = Leptomysis sp., PE = Palaemon elegans, PS = Palaemon serratus.
Left channel
200
Right channel
Time (s)
150
100
50
0 HM
LS
PE
PS
Supplementary Fig. S5. Response of the four crustaceans to chemical cues from control seawater (CS) vs. CS (panels a, d, g and j), sponge conditioned seawater (SCS) vs. CS (panels b, e, h and k), and (c) sponge chemical extracts dissolved in seawater (SCE) vs. CS (panels c, f, i and l). Results are mean time (± SE) in seconds that each species spent in the three compartments of the two-channel flume.
CS vs. CS
Time (s)
250
(a) Hemimysis margalefi
250
200
150
150
150
100
100
100
50
50
50
250
Time (s)
250
200
0 CS
Downstream
CS
(d) Leptomysis sp.
Downstream
CS
(e) Leptomysis sp.
SCE 250
200
200
200
150
150
150
100
100
100
50
50
50
0 CS
250
Downstream
CS
Downstream
CS 250
(h) Palaemon elegans
200
200
150
150
150
100
100
100
50
50
50
250
Downstream
CS
SCS 250
(j) Palaemon serratus
Downstream
250
(k) Palaemon serratus
200
200
150
150
150
100
100
100
50
50
50
Downstream
CS
Downstream
CS
(l) Palaemon serratus
0
0 CS
CS
(i) Palaemon elegans
SCE
CS
200
0
Downstream
0
0 CS
CS
(f) Leptomysis sp.
SCE
200
0
Downstream
0 SCS
250
(g) Palaemon elegans
(c) Hemimysis margalefi
0 SCS
250
0
Time (s)
(b) Hemimysis margalefi
200
0
Time (s)
SCE vs. CS
SCS vs. CS
SCS
Downstream
CS
SCE
Downstream
CS
Supplementary Fig. S6. Heatmap based on the variable importance in projection of the features (VIP > 1) detected in the particulate phase of the seawater samples analyzed by liquid chromatography coupled to mass spectrometry in positive mode. X-axis displays the different seawater treatments, Y-axis displays metabolite features coupling nominal mass (M as atomic mass unit) with chromatographic retention time (T in seconds). Connections of samples and metabolic features are based on hierarchical clustering (Euclidian distances). CS: control seawater; +CS: cave seawater; SCS: sponge conditioned seawater; SCE: sponge chemical extract; p: particulate phase of seawater samples.
Supplementary Fig. S7. Heatmap based on the variable importance in projection of the features (VIP > 1) detected in the dissolved phase of the seawater samples analyzed by liquid chromatography coupled to mass spectrometry in negative mode. X-axis displays the different seawater treatments, Y-axis displays metabolite features coupling nominal mass (M as atomic mass unit) with chromatographic retention time (T in seconds). Connections of samples and metabolic features are based on hierarchical clustering (Euclidian distances). CS: control seawater; +CS: cave seawater; SCS: sponge conditioned seawater; SCE: sponge chemical extract; d: dissolved phase of seawater samples.
Supplementary Fig. S8. Heatmap based on the variable importance in projection of the features (VIP > 1) detected in the particulate phase of the seawater samples analyzed by liquid chromatography coupled to mass spectrometry in negative mode. X-axis displays the different seawater treatments, Y-axis displays metabolite features coupling nominal mass (M as atomic mass unit) with chromatographic retention time (T in seconds). Connections of samples and metabolic features are based on hierarchical clustering (Euclidian distances). CS: control seawater; +CS: cave seawater; SCS: sponge conditioned seawater; SCE: sponge chemical extract; p: particulate phase of seawater samples.
Supplementary Fig. S9. Representative fingerprints of a dissolved phase of a sponge conditioned seawater (SCS) analyzed in (a) negative and (b) positive modes by liquid chromatography coupled to mass spectrometry (base peak chromatogram).
Supplementary Fig. S10. Extracted ion chromatograms for (a) aeroplysinin-1 (m/z 335.8871, [M-H]-, dissolved phase) and (b) aerothionin (m/z 812.8406, [M-H]-, particulate phase) with respective experimental and theoretical mass spectra.
Supplementary Table S1. Post-hoc permutational pairwise tests (999 permutations, model: PPLS-DA, P-value adjustment method: fdr). Significant P-values (< 0.05) are indicated in bold. +CS: cave seawater; SCS: sponge conditioned seawater; SCE: sponge chemical extract.
Dissolved phase
Negative mode
Positive mode
Particulate phase
Negative mode
Positive mode
CS SCE SCS CS SCE SCS CS SCE SCS CS SCE SCS
+CS 0.010 0.010 0.010 0.008 0.008 0.008 0.009 0.009 0.010 0.012 0.006 0.006
CS 0.044 0.083 0.034 0.026 0.032 0.044 0.041 0.671
SCE 0.044 0.031 0.028 0.040
Supplementary Table S2. Tentative annotation of the chemical features detected in the dissolved phases of +CS, SCS and SCE seawater samples analysed by liquid chromatography coupled to mass spectrometry (positive mode). Their selection was based on features with variable importance in projection above 1 (see Fig. 6). The first group of features gathers ions present in +CS and SCE, the 2nd group of features gathers ions present in SCS and SCE. mSigma are given as a quality assignation index based on mass defect and isotopic pattern comparisons with theoretical raw formula. The lower the mSigma value, the better the assignation. +CS: cave seawater; SCS: sponge conditioned seawater; SCE: sponge chemical extract.
Group
Metabolites
Ion
Assignation
1st
A B
M453T323 M475T323 M566T337
C
D
2 nd
Ion monoisotopic mass
Ion formula
Error (mDa)
Error (ppm)
mSigma
Molecular formula
[M+H]+ [M+Na]+ [M+H]+
Intensity ratio (relative to base peak) / counts 100 / 40188 100 / 41780 100 / 110714
453.3435 475.3254 566.4279
[M+H]+
51 / 88180
679.5121
M701T346 M397T352
[M+Na]+ [M+2H]2+
35 / 59555 100 / 172447
701.4940 396.8015
E F G H I
M349T387 M295T419 M331T421 M243T429 M435T438 M437T437
[M+H]+ [M+H]+ [M+H]+ [M+H]+ [M+H]+ [M+H]+
90 / 897 29 / 4893 100 /16726 6 / 2522 100 / 1963
349.2583 295.2264 331.2479 243.1956 435.1467 437.1446
J K
M385T483 M349T486 M350T482 M315T389 M353T440 M330T484
[M+H]+ [M+H]+ [M+H]+ [M+H]+ [M+H]+ [M+H]+
11 / 2357 100 / 5666 66 / 5471 100 / 2815 69 / 18009 100 / 57199
385.1905 349.2141 349.2139 315.2644 353.2297 330.2638
0.1 0.0 1.1 0.3 0.3 1.1 0.3 0.7 0.1 0.6 0.4 0.2 0.3 0.1 0.8 0.1 0.8 0.4 0.0 0.1 0.1 0.2 0.4
0.2 0.0 1.9 -0.5 -0.4 1.5 -0.5 1.8 0.1 -1.5 -1.1 -0.6 0.8 -0.5 1.9 -0.2 1.8 -0.9 0.1 0.3 0.3 -0.5 -1.3
21.1 14.8 2.0 10.0 5.2 6.9 19.7 11.1 17.8 28.0 66.4 6.5 9.5 18.5 56.6 62.4 107.3 41.6 42.3 19.7 39.9 5.5 8.3
C24H44N4O4
M680T345
C24H45N4O4 C24H44N4NaO4 C31H52N9O C30H56N5O5 C36H67N6O6 C37H63N10O2 C36H66N6NaO6 C43H75N11O3 C42H79N7O7 C41H83N3O11 C18H37O6 C18H31O3 C18H35O5 C14H27O3 C18H32BrN2O5 C19H30Cl3N4O C24H29Cl2O3 C18H35Cl2O4 C18H34ClO4 C18H34ClO4 C17H35N2O3 C16H29N6O3 C18H36NO4
L M N
C30H55N5O5 C36H66N6O6
C42H77N7O7
C18H36O6 C18H30O3 C18H34O5 C14H26O3 C18H31BrN2O5
C18H34Cl2O4 C18H33ClO4 C17H34N2O3 C16H28N6O3 C18H35NO4
Supplementary method
Metabolomic analyses The seawater samples were filtered successively through 0.45 µm pore-sized nylon filters at a very low flow using a manual pump to collect the particulate phase. Remaining filtrates were passed through octadecyl-bonded silica extraction discs (EmporeTM C18 SPE discs) under vacuum to extract dissolved metabolites. For the particulate phase, each nylon filter was placed in a 20 mL glass vial with 10 mL of methanol (Sigma-Aldrich) and was sonicated (35 kHz) for 5 min. The filter was then removed from the vial and discarded while the solvent was evaporated until dryness using a SpeedVacTM (Thermosavant, SPD111V). The vial content was solubilized in 1 mL of MeOH and stored refrigerated (4 °C) for 10 h to precipitate salts. Before the filtration of the dissolved phase, the C18 disc was conditioned using the following procedure: the disc was washed with 10 mL of MeOH under vacuum with a pump and dried at higher vacuum for 1 min. The disc was then conditioned with two successive volumes of 10 mL of MeOH at very low vacuum and kept wet until the sample was deposited. Samples were filtered on the extraction disc under vacuum. After filtration, the disc was dried under high vacuum for 5 min to reach complete dryness and metabolites were eluted initially with 10 mL of MeOH. This elution step was then repeated twice with 5 mL of MeOH. The solvent containing the total organic extract was evaporated to dryness using SpeedVac, after which the vial content was solubilized in 1 mL of MeOH and stored refrigerated (4 °C) for 10 h to induce salt precipitation. The supernatant of each extract (i.e. particulate and dissolved phases) was transferred to 10 mL vials and evaporated to dryness using SpeedVac. All extracts were then solubilized in 7 mL of MeOH. They were subsequently filtered through 0.2 µm PTFE filters (Restek) for UHPLC-HRMS analyses. Finally, 500 µL of the filtered extract was transferred into a 2 mL vial and diluted with 500 µL of MeOH in preparation for UHPLC-HRMS analyses.
UHPLC separation was achieved on an analytical Kinetec® Phenylhexyl column (150 × 2 mm, 1.7 µm, Thermo Scientific) using a linear elution gradient of water: acetonitrile (FMP reagents) with 10 mM of ammonium formate in negative mode and 0.1 % formic acid in positive mode from 90:10 (v:v, isocratic from 0 to 2 min) to 0:100 (v:v, isocratic from 8 to 12 min) at a 0.5 mL.min-1 flow rate for a total runtime of 14 min. The injected volume was set to 5 µL. The mass spectrometer analyser parameters were set as follows: nebulizer sheath gas, N2 (2.1 bar); dry gas, N2 (8 L.min-1); capillary temperature, 200 °C; capillary voltage, 2500 V in positive mode and 3000 V in negative mode; end plate offset, 500 V; collision gas, N2; collision energy, 4 eV. Data were acquired at 2 Hz in the 50 to 1200 m/z range in full scan mode.
