Efficiency of Peptide Nucleic Acid-Directed PCR Clamping and Its Application in the Investigation of Natural Diets of the Japanese Eel Leptocephali
et al. (2011) Efficiency of Peptide Nucleic Acid-Directed PCR Clamping and Its Application in the
Investigation of Natural Diets of the Japanese Eel Leptocephali. PLoS ONE 6(11): e25715. doi:10.1371/journal.pone.0025715
Efficiency of Peptide Nucleic Acid-Directed PCR Clamping and Its Application in the Investigation of Natural Diets of the Japanese Eel Leptocephali
Takeshi Terahara 0
Seinen Chow 0
Hiroaki Kurogi 0
Sun-Hee Lee 0
Katsumi Tsukamoto 0
Noritaka Mochioka 0
Hideki Tanaka 0
Haruko Takeyama 0
Brett Neilan, University of New South Wales, Australia
0 1 Department of Life Science and Medical Bioscience, Waseda University, Shinjuku-ku, Tokyo, Japan, 2 National Research Institute of Aquaculture, Yokosuka, Japan, 3 Dong-A University , Saha-gu, Busan , Korea , 4 Atmosphere and Ocean Research Institute, The University of Tokyo, Chiba, Japan, 5 Kyushu University , Fukuoka , Japan , 6 National Research Institute of Aquaculture , Mie , Japan
Polymerase chain reaction (PCR)-clamping using blocking primer and DNA-analogs, such as peptide nucleotide acid (PNA), may be used to selectively amplify target DNA for molecular diet analysis. We investigated PCR-clamping efficiency by studying PNA position and mismatch with complementary DNA by designing PNAs at five different positions on the nuclear rDNA internal transcribed spacer 1 of the Japanese eel Anguilla japonica in association with intra-specific nucleotide substitutions. All five PNAs were observed to efficiently inhibit amplification of a fully complementary DNA template. One mismatch between PNA and template DNA inhibited amplification of the template DNA, while two or more mismatches did not. DNA samples extracted from dorsal muscle and intestine of eight wild-caught leptochephalus larvae were subjected to this analysis, followed by cloning, nucleotide sequence analysis, and database homology search. Among 12 sequence types obtained from the intestine sample, six were identified as fungi. No sequence similarities were found in the database for the remaining six types, which were not related to one another. These results, in conjunction with our laboratory observations on larval feeding, suggest that eel leptocephali may not be dependent upon living plankton for their food source.
Funding: This study was supported by the Ministry of Agriculture, Forestry and Fisheries of Japan and a Grant-in-Aid for Scientific Research on Priority Area (B),
No. 15380137, from the Ministry of Education, Science, Sports and Culture. The funders had no role in study design, data collection and analysis, decision to
publish, or preparation of the manuscript.
Competing Interests: The authors have declared that no competing interests exist.
DNA-based analysis has become a popular tool for molecular
diet analysis [1,2,3,4,5,6]. An orthologous DNA region from a
wide variety of organisms can be amplified by polymerase chain
reaction (PCR) with universal primers, which are subsequently
subjected to nucleotide sequence analysis followed by homology
search. Because PCR usually favors amplification of dominant
DNA molecules, accurate molecular diet analysis is difficult due to
host organism contamination. For example, when stomach
content and host tissue cannot be well separated, as in the case
of an invertebrate or their larvae, the crude DNA preparations
may contain a substantial amount of host DNA. Attempts to
determine prey organisms of the phyllosoma larvae of spiny and
scyllarid lobsters were performed [2,4], in which 18S rDNA
molecules were amplified using a crude DNA template extracted
from the hepatopancreas, cloned, and subjected to restriction
fragment length polymorphism analysis to select clones showing
non-host restriction patterns. They found nearly 90% of the 2,341
clones examined were of the host lobster larvae, indicating that a
substantial amount of contamination from the host genome
PCR-clamping using blocking primer and DNA-analogs, such
as peptide nucleotide acid (PNA) and locked nucleotide acid, may
be a promising technique to inhibit amplification of excess
nontarget DNA [5,6,7]. Using universal primers to amplify ribosomal
DNA internal transcribed spacer 1 (ITS1) with spiny lobster (the
genus Panulirus) specific PNA, a remarkable PCR-clamping of the
host DNA and detection efficiency of the prey DNA was
demonstrated . However, the positional relationship between
PNA and PCR primers, and mismatch between PNA and
template DNA in relation to the clamping efficiency, was not
We have applied the PCR-clamping technique using PNA to
investigate the natural diet of leptocephalus larvae of the Japanese
eel, Anguilla japonica. The natural diet of the eel leptocephali has
been a mystery  and this information may prove important for
developing better artificial food in aquaculture. To design
eelspecific PNA, we determined the ITS1 sequence of the Japanese
eel and found several intra- and inter-individual nucleotide
substitutions. In this study, using the eel ITS1 variants, we
investigated the effects of PNA position and mismatch between
PNA probes and the target sequence. We also report results of our
preliminary attempt to investigate the natural diet of eel
leptocephali using PCR clamping.
All animal sampling in this study complied with the Council of
the European Communities Directive 86/609/EEC regarding the
protection of animals used for experimental and other scientific
purposes, and fully complied with local fisheries management and
marine protected area controls. Wild adult and larval eels
captured with trawl nets deployed from research vessels were
dead on retrieval and sampled at that time.
Samples and DNA extraction
Four adults (eel00, eel01, eel02, and eel03) of the Japanese eel
(Anguilla japonica) were used in this study; two (eel00 and eel01)
were cultured individuals and the others (eel02 and eel03) were
caught in the southern part of the West Mariana Ridge . These
adult samples were used to investigate intra-specific variation in
the ITS1 region. Early stage leptocephalus larvae (c.a. 1017 mm
total length) of the Japanese eel were caught in 2009 and 2010 by
RV Kaiyo Maru and Shoyo Maru (Fisheries Agency, Japan) using
an Isaacs-Kidd Midwater Trawl net at the southern part of the
West Mariana Ridge. Four larvae (designated as 163, 165, 167
and 177) in 2009 and four (designated as St23-10, St23-11,
St2412 and St30-15) in 2010 were rinsed with sterile sea water on
board, fixed in 70% ethanol, and transferred to the laboratory.
Prior to dissection in the laboratory, the body surface of the
leptocephali was rinsed with 70% ethanol. The dorsal muscle of
the larvae was first dissected using sterile forceps and used as a
control. Subsequently, the intestine was dissected and used for diet
analysis. Crude DNA was extracted from these adult and larval
samples using DNeasy Blood and Tissue Kits (Qiagen, Tokyo,
Japan). A DNA sample of the Japanese spiny lobster (Panulirus
japonicus) derived from our laboratory stock was used as a model
Nucleotide sequences of the PCR primers used are shown in
Table 1. SP-1-5 and 5.8S were used to amplify the long fragment of
one adult (eel00). ITS1 of the other adults was amplified using ITS5
and 5.8S. PCR was performed in a reaction mixture (total 25 ml)
containing 26GC buffer I (Takara-Bio), 200 mM
deoxyribonucleotide triphosphate, 0.3 mM each of the universal primers, 0.625 U
of LA Taq DNA polymerase (Takara-Bio), and template DNA.
Amplification was performed with the following profile: 5 min at
94uC; 30 cycles of 1 min at 94uC, 3 min at 53uC, and 7 min of final
extension at 53uC. PCR products were purified with the Wizard SV
Gel and PCR Clean-up System (Promega, Tokyo, Japan), cloned
into pGEM-T easy vector (Promega), and transformed into ECOS
competent E. coli JM109 cells (NIPPONGENE, Tokyo, Japan).
Using M13F and M13R primers, colony direct nucleotide
sequencing was performed to the transformants. The sequence
data were collected on an ABI3130 Genetic Analyzer, assembled,
and analyzed by Bioedit (http://www.mbio.ncsu.edu/RNaseP/
PNA-directed PCR clamping and diet analysis
PNA probes were designed based on the 18S rDNA and ITS1
sequences obtained in this study. Melting temperature (Tm) of
PNA probe was estimated using a calculation tool (http://www.
investigate the effects of PNA position and mismatches between
PNA probes and the target sequence, PNA-directed PCR
clamping was applied to four clones from two adult eels (eel00
and eel01) using 18SL and 5.8S primers under the same conditions
mentioned above, with the addition of 4 mM of PNA probe. These
four clones had 0 to three nucleotide mismatches with PNAs as
described later. To confirm amplification, PCR products were
electrophoresed on 1.5% (w/v) agarose gels followed by 20 min of
staining with ethidium bromide.
For diet analysis, PNA-directed PCR clamping was applied to
eight eel larvae using ITS5 and 5.8S primers under the same
reaction conditions. When no apparent amplification of the eel
ITS1 was observed, clamping was determined to be successful.
The primary PCR products from the DNA templates extracted
from eel larvae dorsal muscle and intestine were subjected to
seminested second PCR using 18S2 and 5.8S primers. PCR products
from the second PCR were cloned and sequenced as described
above. ITS1 sequences obtained were subjected to homology
search using a Basic Local Alignment Search Tool (BLAST) in
GenBank. Because cross contamination from the larval body
surface was unavoidable, we used following criteria for the
interpretation: 1) sequences detected from the dorsal muscle alone
or both dorsal muscle and intestine may not be from intestine, 2)
sequences detected only from intestine may be from intestine, and
3) similar sequences shared by several larvae may be from prey
candidates if the eel larvae are dependent upon certain organisms
Eel ITS1 sequence and design of PNA probes
The nucleotide sequence for a clone (eel00-1) obtained from an
adult individual (eel00) is shown in Fig. 1, and has been deposited
in the DNA Data Bank of Japan under accession No. of
AB617806. This sequence is comprised of 202 bp partial sequence
of 18S rDNA, 422 bp entire sequence of ITS1, and 52 bp partial
sequence of 5.8S rDNA. The ITS1 region is characteristic of high
GC content (69.9%) DNA. Nucleotide sequences of 100 clones
obtained from the other three adults (eel01 to eel03, 30 to 40
clones each) were determined. Four to five ITS1 variants were
observed in each individual, and 11 variants were determined in
total. One variant was the most common and nearly identical to
eel00-1, and the sequences of the other 10 variant types were
deposited in the DNA Data Bank of Japan (AB617807 to
AB617816). Cloning efficiency indicated that the variant types
were minor intragenomic variants or pseudogenes. The length of
ITS1 in these variant types were longer (431 to 437 bp) and the
GC content less (60.7 to 61.4%) than those of the common type
Figure 1. Nucleotide sequence of a 676 bp fragment from a common type clone (eel00-01) amplified from an adult Japanese eel
(Anguilla japonica; eel00). This clone comprised 202 bp partial sequence of 18S rDNA, 422 bp entire sequence of ITS1, and 52 bp partial sequence
of 5.8S rDNA. PCR primers are underlined.
(eel00-1). The nucleotide sequence difference (all substitutions
including indel were equally treated) between variant types and the
common type was 16.261.7%, much larger than those within a
variant type (1.860.4%) and within the common type (0.360.2%).
Figure 2 shows the aligned nucleotide sequences of eel00-1 and
three intra-individual variants from one adult (eel01); the flanking
regions (18S rDNA-ITS1 and ITS1-5.8S rDNA) and positions of
four PNA probes (PNA-F0 to F2 and R1) are designated. The
nucleotide sequences and Tm of PNA probes are presented in
Table 2. All PNA but one (PNA-F3) were a perfect match with the
common type. PNA-F0 (19 bases) and PNA-F3 (17 bases), located
in the flanking region between 18S rDNA and ITS1, shared eight
and three nucleotides with the 18SL primer, respectively. PNA-F0
had one mismatch with the variant types. PNA-F3 (17 bases) had
one mismatch with the common type and two with the variant
types. PNA-F1 and PNA-R1 (17 and 18 bases, respectively) were
designed to be near the 59 end and 39 end of ITS1, respectively,
and had three mismatches, including gaps, with variant types.
PNA-F2 was a perfect match with all types. The Tm of the PNA
probes varied from 67.3uC (PNA-F2) to 86.7uC (PNA-F1).
PCR clamping efficiency in relation to mismatch and
position of PNA
The ITS1 regions of all template DNAs of the common type
(eel00-1) and variant type clones (eel01-01 to eel01-03) of the
Japanese eel and the Japanese spiny lobster were confirmed to be
effectively amplified by PCR primer pairs (18SL and 5.8S). DNAs
of the common type clone (eel00-1) and spiny lobster were mixed
at a ratio of 100:1, and the 100 pmol mixed DNA was used for
PCR using the 18SL and 5.8S primers with and without
eelspecific PNA probes. The result of the PCR clamping is shown in
Fig. 3. Without PNA probe, eel ITS1 (c.a. 500 bp) was
predominately amplified but lobster ITS1 was not (Fig. 3, 2nd
lane from the left). It appeared that the PCR favored dominant
DNA molecules and shorter fragments for amplification.
Amplification of the common eel ITS1 type (eel00-01) was successfully
inhibited by all PNA probes (Fig. 3, 3rd to 6th lane from the left),
although one PNA (PNA-F3) had one mismatch with the common
type (not shown). When amplification of eel ITS1 was effectively
inhibited, the lobster ITS1 (c.a. 680 bp) was amplified (Fig. 3, 3rd
to 6th lane from the left). One clone (eel01-01) of the variant types
was used to investigate the effect of mismatch between the PNA
probes and template DNA (Fig. 4). PNA-F0 (one mismatch)
successfully inhibited amplification of eel ITS1 but did not disturb
amplification of lobster ITS1 (Fig. 4, 3rd lane from the left), while
eel ITS1 was predominately amplified with PNA-F3 (two
mismatch), PNA-F1 (three mismatch), and PNA-R1 (three
mismatch; Fig. 4, 4th to 6th lane from the left).
Investigation of the natural diet of leptocephali
We used PNA-F0 to investigate the natural diet of the
leptocephali. In all samples tested (dorsal muscle and intestine of
eight larvae), no apparent amplification of the eel ITS1 was
observed in the first and second rounds of PCR with PNA-F0.
Forty-eight transformed colonies per sample were subjected to
nucleotide sequence analysis using the M13 forward primer. By
omitting non-ITS1 sequences and clones having no inserts, we
obtained a total of 743 sequences (374 from dorsal muscle and 369
from intestine). Sequence alignments allowed us to classify the 743
sequences into 32 types (lep1 to 33; lep23 not included) and 49
sub-types (Table 3; GenBank accession no. AB616868-AB616909,
AB616911-AB616920). We found very similar or nearly identical
sequences in the database for 19 types (lep14 to 33), where the
homology score between the query sequences and the highest
similar sequences ranged from 85 to 99%. Among them, 17 types
appeared to be fungi (lep16 to 33). The length of these fungal ITS1
sequences varied from 58 to 267 bp and GC content varied from
23.7 to 64.1%. Among fungal sequences, lep16 (Malassezia spp.)
was detected in both dorsal and intestine samples of almost all
larvae. One type (lep14) detected from dorsal muscle and intestine
of a larva (St24-12) was identical to a macroalgal species
(Eckloniopsis radicosa), and another (lep15) obtained from an
intestine sample of a larva (St23-11) was identical to an
undetermined eukaryote (AB490762). No other sequence identities
were found in the database for the remaining 13 types (lep1 to 13),
and these types were not related one another. According to the
*Nucleotide sequences of all sub-types are available in DNA database (AB616868-AB616909, AB616911-AB616920).
1Larval strains named after sampling station.
above-mentioned criteria, 12 types (lep2, 6, 810, 15, 18, 21, 24,
25, 27, and 30) may be from intestine and only one (lep21) was
shared by different larvae.
PNA directed PCR-clamping
All PNA probes successfully inhibited amplification of fully
complementary eel ITS1, indicating that position of the PNA has
little effect on clamping as long as the Tm is higher than that of the
PCR primers. Two or more base pair mismatches between PNA
probes and the template sequences were sufficient to dramatically
reduce the clamping efficiency (Fig. 4), although designing PNA
having higher Tm may improve clamping efficiency. A single
mismatch is known to considerably decrease the Tm of PNA probe
at 820uC . Unsuccessful clamping using PNA-F3 and
variant types indicated that the two bp mismatch lowered the Tm
of the PNA below that of the primers. Igloi  using 11-mer PNA
and its complementary DNA demonstrated the importance of the
position of the DNA/PNA mismatch and mismatch types as well.
Igloi  observed that a single mismatch positioned at the center
or terminus of the PNA/DNA duplex was more stable than that
positioned at 4 bases from terminus and that stabilization was
maximum for G:T and T:T mismatched pairs and minimum for
A:A and G:G pairs. In our study, PNA-F0 had one mismatch
(C:C) with the variant types at terminus and PNA-F3 had one
mismatch (G:T) with the common type at 5 bases from terminus.
Both PNAs successfully inhibited amplification of variant and
common types, respectively, suggesting that the Tm of these PNA
were higher than that of the primers in spite of the presence of one
Slight difference in amplification efficiency may be observed
among PNAs used (see Fig. 3). Although the difference may be
caused by loading amount and/or leakage of DNA samples in
wells, non-specific PCR clamping can not be ruled out. PNA-F0
was designed in the flanking region between 18S rDNA and ITS1.
The upper eight nucleotides are at 39 end of 18S rDNA and
universal, and subsequent three nucleotides are at 59 end of ITS1
and semi-universal , which may elevate stabilization of the PNA
on non-specific DNA templates.
Investigation of natural diet of the leptocephali
The efficiency of PNA-directed PCR clamping adopted in the
present study was remarkable, in that we found no clone
containing eel ITS1 sequence among 743 clones examined. More
than half of the sequence types obtained appeared to be of fungi,
with the genus Malassezia extensively detected in our samples. This
fungal genus comprises a group of superficial fungi occurring as
skin flora on the human and animal body, but not in the
environment . Furthermore, only two (lep 19 and 28) among
17 fungal types detected are seen in the complete list of higher
marine fungi (http://ocean.otr.usm.edu/,w529014/index_files/
Page1195.htm). Therefore, many fungal strains detected in the
present study may be the result of cross contamination during the
handling process on the research vessel and/or in the laboratory.
By omitting fungi, six types (lep 2, 6, 810, and 15) were thought
to be from the intestine. No sequence similarity among them was
observed, indicating that the leptocephali may not be dependent
on a narrow range of organisms for their food source.
The variation in ITS1 length we observed ranged from 58 to
391 bp (Table 3). In contrast to the coding region of rDNA, large
ITS1 length variations have been observed, even within closely
related taxa. Variation in length of fungal ITS1 has been observed
ranging from 140 to 1,100 bp [14,15] and extreme length
variation (791 to 2,572 bp) has been observed in ladybird beetles
. In marine animals, vertebrate ITS1 (Osteichthyes and
Chondrichthyes) is relatively long (318 to 2,318 bp) compared
with that of invertebrates (117 to 1,613 bp), and ITS1 of
gelatinous animals (Cnidaria and Ctenophora) is especially short
(118 to 422 bp) . Amplification efficiency may be considerably
different between shorter and longer fragments .
ITS1 sequences obtained in the present study were confined to
the shorter range, suggesting that the detection of eukaryotes
having shorter ITS1 was biased or that eel leptocephali consume
eukaryotes having shorter ITS1. The results obtained in this study
may correspond to a recent study of the diet of European eel (A.
anguilla) larvae using 18S rDNA separated by denaturing gradient
gel electrophoresis (DGGE) , which suggested that gelatinous
zooplankton is common prey for the eel larvae. Riemann et al.
 proposed that they detected a wide variety of animal phyla,
including fungi and phytoplankton, from the larval eel gut;
however, they did not analyze a control free from gut contents,
such as dorsal muscle.
An alternative hypothesis is that eel leptocephali consume
already-degraded material, which is consistent with information of
Pfeiler  and Otake et al.  that eel leptocephali use dissolved
organic matter and/or particulate organic matter (POM). Longer
fragments may be more susceptible to digestion than shorter
fragments . On the other hand, oikopleurlid larvacean houses
and zooplankton fecal pellets were observed in the gut of
leptocephali of eight eel species, and were considered a major
dietary component for leptocephalus larvae . Furthermore,
aloricate protozoa were considered to be a significant energy
source for eel leptocephali . However, wild-caught
leptocephali of the conger eel Conger myriaster and cultured Japanese eel
leptocephali were not attracted to Oikopleura dioica fed in the
laboratory, even when the leptocephali contacted the larvacea (Dr.
Kurogi, personal communication). Cultured leptocephali of the
Japanese eel showed no active feeding behavior toward
zooplankton . Wild-caught leptocephali of the pike eel Muraenesox
cinereus and conger eel C. myriaster were observed ingesting and
defecating squid paste . A slurry-type diet made from shark
egg yolk powder was found to be a suitable food for captive-bred
Japanese eel larvae , and the first cultivation of the glass eel in
the laboratory was established by this diet supplemented with krill
hydrolysate, soybean peptide, vitamins, and minerals .
Although the Japanese eel actively engulfs the slurry-type diet,
they do so only when they contact the food (Dr. Masuda, personal
communication). Otherwise, the food was unnoticed by
leptocephali, indicating that, unlike other fish larvae, leptocephali do
not seem to depend on visual and olfactory sensing for food. Based
on his comprehensive review of leptocephalus feeding ecology,
Miller  seemed fairly convinced that marine snow-like material
is the major food for leptocephali. The present study detected no
specific DNA common to leptocephalus larvae, and previous
studies reported a wide variety of small organisms, larvacean
houses, and fecal pellets as food [23,24]. This corresponds to
Millers implication and is further corroborated by stable isotope
ratio analysis on the eel leptocephali and POM [21,28].
The authors thank crew members of the R/V Kaiyo Maru for supporting
sample collection. We also thank Mayumi Sato, Tomoko Kawashima, and
Chie Takahashi for assisting with DNA analysis and K. Hatamura for
Conceived and designed the experiments: H. Takeyama SC TT.
Performed the experiments: TT SC. Analyzed the data: TT SC H.
Takeyama SHL. Contributed reagents/materials/analysis tools: SC HK
KT NM H. Tanaka. Wrote the paper: TT SC H. Takeyama.
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