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 (-80C) 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
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