Supporting Online Material, Riemann et al. 1. 2. 3. 4. 5. Supporting Materials and methods Supporting text Supporting figures S1 and S2 Supporting tables S1 – S3 Supporting references 1. Materials and methods Capture of eel larvae. At each station fish larvae were sampled using a ring net of 3.5 m diameter equipped with a 25 m long net of 600 µm mesh. This gear was lowered to 250 m in an oblique haul at a speed of 2.5 knots. The catch was brought to the laboratory, kept cold, and within 1 h, Anguilla-like larvae were sorted, photographed, had their length measured and were preserved in RNAlaterTM (QIAGEN). The remaining sample was preserved in 96% ethanol. After the cruise some additional larvae (A. anguilla and A. rostrata) were selected as described below. To compare potential differential effects of ethanol preservation and freezing, the guts of 16 larvae were excised immediately after sampling (described below) and frozen (-80C) individually in sterile plastic tubes (see supporting text). Molecular identification of Anguilla anguilla larvae. The 16 larvae for which guts were excised onboard were tentatively identified through amplification and sequencing of a ~655 bp region of the Cytochrome c oxidase subunit I (COI) gene (Ward et al. 2005). For the other leptocephali species assignment to A. anguilla was carried out using two different molecular analyses of DNA extracted from each larva (using the E.Z.N.A.TM kit, OMEGA BIOTEK, Norcross, Georgia, USA): 1) PCR was carried out using a mix of four species-specific primers (Trautner 2006), where different primer pairs amplify fragments of the maternally inherited mitochondrial cytochrome b region. Amplicon length differs between A. anguilla (789 bp) and A. rostrata (589 bp). Other non-Anguilla species exhibit different amplicon sizes or do not amplify at all. Amplicon sizes were determined by agarose (1%) gel electrophoresis and ethidium bromide staining. 2) Fragments of the nuclear 5S rRNA gene region were amplified, and amplicon sizes enabled the distinction of A. rostrata (~1200 bp) and A. anguilla (~600 bp) (Pichiri et al. 2006). Finally, as part of a subsequent population genetic analysis, 21 microsatellite loci were analysed, which allowed for double-checking species identification. Bayesian model based clustering of multi-locus genotype data, as implemented in STRUCTURE 2.2 (Pritchard et al. 2000), was used for distinguishing A. anguilla from A. rostrata, whereas non-anguillid larvae typically did not amplify at the majority of the microsatellite loci. Further details on microsatellite analysis are available from the authors on request. Gut excision and molecular analyses of gut contents. Guts were removed under a dissection microscope. A longitudinal cut was made above the gut, which was subsequently excised using aseptic technique and flaming of tools between samples. The guts were stored individually in 96% ethanol until DNA extraction. DNA was extracted using an enzyme/phenol-chloroform protocol (Riemann et al. 2008) from the 16 guts excised during the cruise and from guts from 61 A. anguilla larvae genetically identified after the cruise. DNA concentration was determined using PicoGreen (Molecular Probes, Paisley, UK). 18S rRNA genes were amplified using universal primers (Sogin & Gunderson 1987; Amann et al. 1 1990) as designed for DGGE (Diez et al. 2001). Mixing of Taq Ready-To-Go PCR Beads (GE Healthcare) and primers (Euk1A and Euk516r-GC) were done in a UV treated sterile flow-bench, DNA template (1 – 3 ng per 25 l reaction) was added in a PCR/UV workstation in a separate room (DNA/RNA UV-cleaner UVC/T, Talron Biotech), and single tubes (not strips) were used. Pipettes, tips and water were UV treated and filter tips were used. Negative controls for PCR (UV-treated Milli-Q water instead of template) and for PCR/DNA extraction (extract from only extraction chemicals used as template in the PCR) never showed amplification. Amplicon purity and correct size (560 bp) was confirmed by agarose gel electrophoresis. Clone libraries were generated from two amplified samples according to the manufacturer’s instructions (TOPO TA Cloning, Invitrogen) and 31 clones were sequenced (Macrogen, Korea; see Supporting text). DGGE analysis, band excision and sequencing were done as previously described (Riemann et al. 2008) except that 60 ng (16 larvae; Table S3) or 600 ng amplicons were applied per sample. Time-travel experiments were used to determine the optimal electrophoresis conditions (6 h at 150 V, denaturant gradient of 20 – 50%, 6% acrylamide). Band detection was done using the software Quantity One 4.6.3 (BioRad) after background subtraction using a rolling disk size set to 8. Bands having the same position between different lanes were aligned and matched (tolerance level set to 0.5 %). Among the obtained sequences, 4 potential chimeras (ELD 64, 68, 69, and 78) were detected using the Mallard and Pintail software (Ashelford et al. 2006). 2. Supporting text Additional information on DNA barcoding of gut contents Since we had no a priori knowledge about potentially consumed prey we used “universal” primers targeting a wide range of eukaryotic taxa. We chose to amplify the multi-copy nuclear 18S ribosomal RNA gene because it has been successfully used to differentiate diverse marine plankton taxa (Diez et al. 2001; Martin et al. 2006; Suzuki et al. 2006), the number of available 18S rRNA gene sequences (283,347 entries on 23 March 2010, http://www.ncbi.nlm.nih.gov/sites/entrez, search string: 18S) exceeds that of any other candidate gene in GenBank (e.g., the mitochondrial cytochrome oxidase I gene), and universal primers are available (Diez et al. 2001). In a test clone library (31 sequenced clones) of amplicons from a gut, >90% of the sequences were related to eels. Since analyses of extensive clone libraries from a large number of larvae were not attractive, we chose to separate prey from eel amplicons based on DNA melting temperature in denaturing gradient gel electrophoresis (DGGE). Eel amplicons were consistently seen as a dense band clearly separated from bands representing prey (Fig. S2). This was confirmed by sequencing of five eel bands from separate samples (data not shown). Repeated PCR-DGGE analyses of 10 samples showed that banding patterns for eel and prey amplicons were always reproducible. Assessment of potential DNA contamination problems DNA contamination from external sources is a potential problem when analysing prey items using PCR based methodology. In our study, results from negative controls excluded contamination from extraction chemicals or PCR reagents. An aseptic technique was applied and tools were flamed between samples to minimize the risk of transfer of external DNA potentially associated with the larval surface to the interior gut during dissection. Several lines of evidence suggest that the identified prey items do not originate from contamination. First, potential transfer of surface-bound DNA to the gut would be assumed to be relatively uniform among larvae obtained from the same sample; however, no prey items were detected in 19 of 2 61 A. anguilla larvae guts and different larvae originating from the same plankton sample showed large variability in the number of detected prey items (0 – 17). Second, the mean number of prey items per larvae did not differ significantly (two-tailed t test, p = 0.39) between larvae stored with plankton in ethanol before sorting (n = 61; table S1) and those sorted and frozen immediately after capture (n = 16; Table S2). Hence, storage of eel larvae with other plankton organisms, i.e. supposedly in a solution with dissolved DNA, did not affect the number of prey items detected. Though, it should be noticed that not all of the 16 eel larvae frozen after capture were A. anguilla. Third, in a similar analysis of prey within lobster larvae using universal primers, contamination from surface-associated external DNA was not detected (Suzuki et al. 2006). Fourth, since rare templates in a PCR reaction may not undergo amplification (Morrison & Gannon 1994) and templates representing <1% of the DNA in a PCR reaction mixture are not detectable in DGGE analysis (Muyzer et al. 1993), it is unlikely that a hypothetical contamination with surface-bound trace DNA would generate a discernible signal in our gut analyses. 3 3. Supporting figures 5 mm N 18 ˚C 35 Latitude (˚N) 19˚C Gut 30 ˚C 20 22 ˚C 23 ˚C 24˚C 25 ˚C 25 26˚C 20 80 75 70 65 60 Longitude (˚W) Figure S1. Map showing the transects in the Sargasso Sea with sampled stations from which the analysed A. anguilla larvae were obtained, indicated as white dots. The sea surface isotherms are based on remotely sensed temperature data from April 4, 2007 obtained from the Operational Sea Surface Temperature and Sea Ice Analysis project (OSTIA, http://ghrsstpp.metoffice.com). Closely spaced isotherms indicate fronts. Inserted picture shows an A. anguilla larva with a visible gut. 4 Band code Sequence length (bp) 1 485 2 485 3 472 4 481 5 485 6 483 7 506 8 484 9 484 10 486 11 486 12 483 13 486 14 510 15 526 Nearest GenBank relative Pinus luchuensis Uncultured streptophyte Collozoum pelagicum Neocercomonas sp. Hyphochytrium catenoides Sistotrema brinkmannii Thysanopoda aequalis Thalia democratica Thalia democratica Vogtia glabra Sphaeronectes gracilis Liriope tetraphylla Tomopteris sp. Conchoecia sp. Pseudosagitta lyra Sequence similarity (%) 100 94 94 90 100 96 99 96 94 100 98 99 100 92 94 D1 NA NA NA D2 NA NA NA D3 NA NA NA Higher taxon Streptophyta Streptophyta Polycystinea Cercozoa Stramenopiles Basidiomycota Malacostraca Thaliacea Thaliacea Hydrozoa Hydrozoa Hydrozoa Polychaeta Ostracoda Chaetognatha Hydrozoa Polycystinea Hydrozoa Accession no. D38246 EU647131 AF091146 AY884333 X80344 DQ898712 DQ900735 D14366 D14366 AY937350 AF358070 AF358061 DQ790095 AB076658 DQ351880 NA NA NA Figure S2. Example of denaturing gradient gel electrophoresis (DGGE) fingerprint of 18S rRNA genes amplified from guts of 12 European eel larvae. Identified bands are labelled with number (sequenced bands) or the letter D (deduced from the vertical alignment with sequenced bands). Unlabelled bands could not be sequenced or the identity could not be deduced. Grey boxes encompass amplicons originating from eel DNA. The total number of discernible prey bands is indicated below each lane. The nearest relatives of the obtained band sequences based on BLAST searches in GenBank are listed in the table. The broader taxonomic affiliation is supported by topological position in the phylogenetic tree in Fig. 1. NA: not applicable. The information from all DGGE analyses is summarised in Table S1. 5 4. Supporting tables 2022 1884 1884 1884 2022 1946 1884 2022 2022 2022 1946 1946 1946 1583 1504 1884 2022 2022 2457 1549 1946 1946 1946 2022 2035 1504 1946 1946 2035 2457 1504 1946 1946 1946 2022 1943 1945 1946 2022 1583 1946 1945 1945 1504 1504 1946 1946 1884 1549 1946 1688 2022 2317 1945 2035 2457 1583 1884 1884 2317 1504 D D D D D N D D D D N N N D N D D D D N N N N D N N N N N D N N N N D N N N D D N N N N N N N D N N D D D N N D D D D D N 4.0 4.5 5.0 5.0 5.5 5.5 6.0 6.0 6.0 6.5 7.0 7.0 7.0 7.0 7.0 7.5 7.5 7.5 7.5 7.5 8.0 8.0 8.0 8.0 8.0 8.0 8.5 8.5 8.5 8.5 8.5 9.0 9.0 9.0 9.0 9.0 9.0 9.5 9.5 9.5 10.0 10.0 10.0 10.0 10.0 10.5 10.5 10.5 10.5 11.0 11.0 11.5 11.5 11.5 12.0 12.0 12.0 12.5 14.0 14.5 14.5 Unidentified band(s) Streptophyta Stramenopiles Thaliacea Polycystinea Polychaeta 1 Ostracoda 1 Malacostraca -64.001 -64.000 -64.000 -64.000 -64.001 -63.998 -64.000 -64.001 -64.001 -64.001 -63.998 -63.998 -63.998 -67.003 -69.999 -64.000 -64.001 -64.001 -63.997 -70.001 -63.998 -63.998 -63.998 -64.001 -64.000 -69.999 -63.998 -63.998 -64.000 -63.997 -69.999 -63.998 -63.998 -63.998 -64.001 -67.001 -67.001 -63.998 -64.001 -67.003 -63.998 -67.001 -67.001 -69.999 -69.999 -63.998 -63.998 -64.000 -70.001 -63.998 -70.001 -64.001 -66.998 -66.996 -64.000 -63.997 -67.003 -64.000 -64.000 -66.998 -69.999 Hydrozoa 26.501 26.000 26.000 26.000 26.501 25.254 26.000 26.501 26.501 26.501 25.254 25.254 25.254 24.500 26.000 26.000 26.501 26.501 27.660 27.501 25.254 25.254 25.254 26.501 27.329 26.000 25.254 25.254 27.329 27.660 26.000 25.254 25.254 25.254 26.501 27.749 25.330 25.254 26.501 24.500 25.254 25.330 25.330 26.000 26.000 25.254 25.254 26.000 27.501 25.254 26.499 26.501 26.501 25.664 27.329 27.660 24.500 26.000 26.000 26.501 26.000 Entomophthoromycotina 9 8 8 8 9 7 8 9 9 9 7 7 7 24 27 8 9 9 12 30 7 7 7 9 11 27 7 7 11 12 27 7 7 7 9 17 22 7 9 24 7 22 22 27 27 7 7 8 30 7 28 9 19 21 11 12 24 8 8 19 27 Dinophyceae 1 1 1 1 1 1 1 1 1 1 1 1 1 2 3 1 1 1 1 3 1 1 1 1 1 3 1 1 1 1 3 1 1 1 1 2 2 1 1 2 1 2 2 3 3 1 1 1 3 1 3 1 2 2 1 1 2 1 1 2 3 Length (mm) Ctenophora P035 P025 P026 P027 P036 P001 P028 P037 P038 P039 P005 P006 P007 P078 P099 P030 P041 P042 P053 P120 P009 P010 P012 P043 P049 P105 P013 P015 P050 P054 P107 P016 P017 P018 P044 P058 P068 P019 P045 P080 P020 P069 P070 P102 P103 P021 P022 P032 P124 P024 P114 P046 P062 P066 P052 P055 P081 P033 P034 P063 P097 Phytoplankton biomass (mg C m-2) Day/Night Copepoda Longitude (degrees W) Chaetognatha Latitude (degrees N) Cercozoa Station no. Basidiomycota Transect no. Ascomycota Larvae ID no. Anthozoa Table S1. Summary of gut content analyses of European eel larvae. Taxonomic lineages of prey identified through denaturing gradient gel electrophoresis and band sequencing of 18S rRNA genes (~500 bp) PCR amplified from guts excised from 61 individual European eel larvae. Numbers in blue indicate sequenced bands. Unmarked numbers indicate bands for which identities were deduced (see Materials and Methods). Unidentified bands could not be sequenced or the identities could not be deduced. Note that some guts were empty, as indicated by the lack of visible bands. Phytoplankton biomass was estimated from cell sizes and counts obtained by flow cytometry and microscopy (L. Riemann, unpublished). 2 1 1 1 1 1 3 1 1 1 1 1 1 1 1 1 2 1 2 2 1 1 1 1 2 1 2 1 1 1 1 1 2 1 1 1 1 1 1 1 1 2 1 1 1 1 1 1 2 1 1 1 1 1 1 2 1 1 1 1 2 1 1 1 1 4 1 1 1 1 5 4 4 1 1 2 1 1 2 1 1 1 1 1 1 1 1 1 1 1 3 1 1 1 1 6 1 1 1 2 3 Table S2. The number of different prey per larva (Table S1) appeared to be overdispersed caused by zero inflation and overdispersion of the non-zero count data (results not shown) (Zuur et al. 2010). The effect of phytoplankton biomass, larva length, time of day, latitude and longitude on the number of different prey per larva (Table S1) was therefore tested using a zero-inflated negative binomial mixture model (Zuur et al. 2010). This was conducted using the zeroinfl function of the pscl package in R (R Development Core Team 2009). Neither of the tested explanatory variables appeared to have a significant effect on the number of different prey per larva, although longitude appeared to have a marginal effect on number of prey per larvae in the count model. The lack of significant effects may however be due small sample size and low power. Count model coefficients (negative binomial with log link): Estimate St. Error (Intercept) -10.0241 7.95617 -2 Phytoplankton biomass (mg C m ) 28.21311 100.5977 Length (mm) 0.0454 0.07792 Latitude 0.03318 0.21515 Longitude -0.1342 0.09896 Day Night 0.51595 0.36562 Log(theta) 0.67027 0.55153 Zero-inflation model coefficients (binomial with logit link): Estimate St. Error (Intercept) 13.4070 71.2400 -2 Phytoplankton biomass (mg C m ) -227.5000 1271.0000 Length (mm) -0.5955 0.9453 Latitude -0.2905 1.2730 Longitude 0.0062 1.0580 Day Night 2.3340 3.5240 Log(theta) 13.4070 71.2400 z-value Pr(>|z|) -1.26 0.28 0.583 0.154 -1.356 1.411 1.215 0.208 0.779 0.56 0.877 0.175 0.158 0.224 z-value Pr(>|z|) 0.188 -0.179 -0.63 -0.228 0.006 0.662 0.188 0.851 0.858 0.529 0.82 0.995 0.508 0.851 Theta = 2.0761, Log-likelihood: -113.1 on 13 Df 7 2 17 13 Anguilla anguilla (99) 2 17 12 L146 GU188388 Anguilla rostrata (98) 2 21 15 M1 GU188376 Serrivomer lanceolatoides (99) 2 19 NA M2 GU188377 Nemichthys scolopaceus (98) 2 19 NA M3 GU188378 Anguilla rostrata (99) 2 19 NA M4 GU188379 Derichthys serpentinus (99) 2 20 19 M5 GU188380 Serrivomer lanceolatoides (99) 2 24 NA M8 GU188381 Nemichthys scolopaceus (98) 3 27 42 M11 GU188382 Nemichthys scolopaceus (98) 3 32 65 M7 NA NA 2 24 17 M9 NA NA 3 29 27 M10 NA NA 3 32 58 2 Unidentified bands Anguilla rostrata (99) GU188387 Streptophyta GU188386 L141 Stramenopiles L140 Thaliacea 13 Polycystinea 17 Polychaeta 2 Ostracoda GU188385 Malacostraca L139 Hydrozoa 11 Entomophthoromycotina 12 17 Dinophyceae 17 2 Ctenophora 2 Anguilla anguilla (99) Anguilla anguilla (100) Copepoda Anguilla anguilla (99) GU188384 Chaetognatha GU188383 L138 Cercozoa L137 Basidiomycota Nearest relative Transect Station Length in GenBank (% similarity) no. no. (mm) Ascomycota GenBank acc. no. Anthozoa ID no. Alveolata Table S3. Summary of gut contents analyses of 16 larvae for which the guts were excised onboard ship and frozen. Numbers in blue indicate sequenced bands. Unmarked numbers indicate bands for which identities were deduced (see Materials and methods). Unidentified bands could not be sequenced or the identities could not be deduced. Sixty ng DNA per sample was used in the DGGE analysis. Larvae were identified by amplification and sequencing of the mitochondrial cytochrome oxidase I gene (Ward et al. 2005). NA: not available. Sequences from prey organisms (ELD80 – ELD90, starting from the top of the table) have been deposited in GenBank under the accession numbers: GU188365-GU188375. 2 1 2 1 1 1 1 1 1 1 1 4 1 1 2 2 1 8 5. Supporting references Amann, R. I., Binder, B. J., Olson, R. J., Chisholm, S. W., Devereux, R., and Stahl, D. 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