Determining the Diet of Larvae of Western Rock Lobster

(Panulirus cygnus) Using High-Throughput DNA

Sequencing Techniques
Richard O’Rorke1*, Shane Lavery1,2, Seinen Chow3, Haruko Takeyama4, Peter Tsai2, Lynnath E. Beckley5,
Peter A. Thompson6, Anya M. Waite7, Andrew G. Jeffs1
1 Leigh Marine Laboratory, University of Auckland, Warkworth, New Zealand, 2 School of Biological Sciences, University of Auckland, Auckland, New Zealand, 3 National
Research Institute of Fisheries Science, Yokosuka, Japan, 4 Department of Life Science and Medical Bioscience, Waseda University, Shinjuku-ku, Tokyo, Japan, 5 School of
Environmental Science, Murdoch University, Murdoch, Western Australia, Australia, 6 Australian Commonwealth Scientific Industrial and Research Organisation, Hobart,
Tasmania, Australia, 7 School of Environmental Systems Engineering and the Oceans Institute, University of Western Australia, Crawley, Western Australia, Australia

Abstract
The Western Australian rock lobster fishery has been both a highly productive and sustainable fishery. However, a recent
dramatic and unexplained decline in post-larval recruitment threatens this sustainability. Our lack of knowledge of key
processes in lobster larval ecology, such as their position in the food web, limits our ability to determine what underpins this
decline. The present study uses a high-throughput amplicon sequencing approach on DNA obtained from the
hepatopancreas of larvae to discover significant prey items. Two short regions of the 18S rRNA gene were amplified under
the presence of lobster specific PNA to prevent lobster amplification and to improve prey amplification. In the resulting
sequences either little prey was recovered, indicating that the larval gut was empty, or there was a high number of reads
originating from multiple zooplankton taxa. The most abundant reads included colonial Radiolaria, Thaliacea,
Actinopterygii, Hydrozoa and Sagittoidea, which supports the hypothesis that the larvae feed on multiple groups of
mostly transparent gelatinous zooplankton. This hypothesis has prevailed as it has been tentatively inferred from the
physiology of larvae, captive feeding trials and co-occurrence in situ. However, these prey have not been observed in the
larval gut as traditional microscopic techniques cannot discern between transparent and gelatinous prey items in the gut.
High-throughput amplicon sequencing of gut DNA has enabled us to classify these otherwise undetectable prey. The
dominance of the colonial radiolarians among the gut contents is intriguing in that this group has been historically difficult
to quantify in the water column, which may explain why they have not been connected to larval diet previously. Our results
indicate that a PCR based technique is a very successful approach to identify the most abundant taxa in the natural diet of
lobster larvae.

Citation: O’Rorke R, Lavery S, Chow S, Takeyama H, Tsai P, et al. (2012) Determining the Diet of Larvae of Western Rock Lobster (Panulirus cygnus) Using High-
Throughput DNA Sequencing Techniques. PLoS ONE 7(8): e42757. doi:10.1371/journal.pone.0042757
Editor: Senjie Lin, University of Connecticut, United States of America
Received April 26, 2012; Accepted July 10, 2012; Published August 21, 2012
Copyright: ß 2012 O’Rorke et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits
unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Funding: The experimental component of this project was funded by a grant from the Fisheries Research and Development Council Australia (http://www.frdc.
com.au/) under FRDC PROJECT NUMBER: 2010/047, and ship time was provided by the Australian Marine National Facility under grant SS05-2010 (http://www.
csiro.au/Organisation-Structure/National-Facilities/Marine-National-Facility.aspx). 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.
* E-mail: [email protected]

Introduction phosis into nektonic post-larvae, or pueruli, which actively migrate

back onshore [4]. For P. cygnus pueruli there is good evidence that
Despite considerable research into the biology of spiny lobsters the magnitude of recruitment to the coast of Western Australia is
(Family Palinuridae), the larval phase of their lifecycle remains positively correlated with westerly winds [5], La Niña events, and
enigmatic. In particular, the composition of the diet of larvae the associated increase in strength of the Leeuwin Current [5–7].
remains uncharacterised for all species of spiny lobster [1], However, the underpinning causal details of how these oceanic
including the western rock lobster (Panulirus cygnus), which is the events impact lobster ecology and recruitment to the coastal
basis of the second largest commercial spiny lobster fishery in the benthic stock remain to be fully established.
world [2]. All evidence indicates that the puerulus is a non-feeding phase
Spiny lobsters have an unusually long planktonic larval phase that fuels its shoreward migration by metabolising extensive lipid
and P. cygnus has an estimated larval duration of 9 to 11 months reserves built up during the preceding phyllosoma phase which
that is spent in oceanic waters extending from the continental shelf actively feeds in the pelagic environment [8–10]. For example, the
margin to over 1,500 km offshore from Western Australia [3]. puerulus of P. cygnus caught closer to shore have markedly lower
After this oceanic phase the larvae, which are known as lipid reserves than those caught further offshore [8], a phenom-
phyllosomata (singular: phyllosoma), are thought to be carried enon consistent with observations of the pueruli of other species of
shoreward by ocean currents and eventually undergo metamor- spiny lobster [11,12]. Settled P. cygnus pueruli also display seasonal

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 Larval Lobster Diet

fluctuations in lipid reserves indicating environmental effects on type, one Panulirus japonicus phyllosoma from the Atlantic Ocean
available energy that may be due to increased metabolic activity and two from the Pacific were analysed and returned amplicons
from elevated temperature, having to traverse ocean currents and from the phylum Cnidaria and the sub-phylum Urochordata [26].
meso-scale oceanic features, or seasonal changes in pelagic prey The same study found DNA of Urochordata and Cnidaria in the
availability to phyllosomata [9]. Pueruli that have traversed a gut of one of two slipper lobsters (Scyllaridae) that were also
greater distance to settle on the coast tend to have more depleted analysed [26]. A subsequent study used the same methods on
lipid reserves than those that have travelled shorter distances, but eleven phyllosomata and detected DNA from teleost fish in three
this pattern is not consistent [9]. The inconsistency could be due to larvae [28]. However, these PCR reactions generated an
final stage phyllosomata undergoing metamorphosis without overwhelming quantity of lobster (host) PCR amplicons and
sufficient lipid reserves to migrate successfully to shore. This numerous clones had to be screened prior to sequencing. The
hypothesis is supported by observations of the red rock lobster, problem of host derived amplicons swamping the prey amplicons
Jasus edwardsii, for which 16.5% of nektonic pueruli were estimated was significantly reduced in a subsequent study, that utilised a
to have insufficient lipid to reach settlement sites on the coast [13]. lobster specific peptide nucleic acid clamp (PNA-clamp) [29], a
Likewise, estimates based on the biomechanics of swimming by method that selectively blocks predator PCR amplification and
spiny lobster pueruli confirm that the lipid energy reserves are therefore enriches prey signal. Although such PCR enrichment
marginal for ensuring settlement success [14]. Puerulus mortality techniques have been common in medical contexts for over two
due to exhaustion of lipid reserves is presumably more of a risk in decades, they are only a recent innovation in ecological studies
P. cygnus, which has significantly smaller larvae and post-larvae and [30–33]. Another improvement was to target a shorter genomic
therefore a reduced ‘‘capacity to store lipid’’ [8]. Any hypothesis region [29], an approach that is consistent with the consensus
based on the feeding of phyllosomata and their nutritional status at approach to DNA diet studies [34,35]. By using PNA-clamping,
metamorphosis is contentious and, currently, not experimentally ten out of thirty-nine phyllosomata were found to contain DNA
testable because it is neither known what the prey of phyllosomata from the phyla Cnidaria, Ctenophora, Arthropoda, Vertebrata
are, nor what ‘triggers’ metamorphosis [4,15]. The present study (Teleostei) and Chaetognatha [29].
examines the efficacy of a molecular approach to identify the prey Despite the success of these DNA-based dietary studies, their
so that future studies can assess the impact of oceanographic authors have indicated several shortcomings that require addi-
events on prey abundance and health. This would be timely tional innovations to enable an efficient DNA-based method to
because a recent collapse in puerulus recruitment to the Western study the diet of phyllosomata. In particular, their results
Australia coast has dramatically impacted the fishery [16], which contained a high occurrence of PCR sequences that are unlikely
has financial as well as ecological implications. Rock lobsters are to be credible prey sequences [26,28,29]. Such sequences included
Australia’s most valuable fishery and Western Rock lobsters host sequence variants (either PCR artefacts or pseudogenes),
contributed over 60% of the catch up to the 2003–2004 financial chimeras and also a high percentage of amplicons derived from
year, but this contribution has trended down to 50% for 2009– microscopic eukaryotes with a questionable role in phyllosoma
2010 [2]. Following this trend the total export value of rock nutrition [28,29]. Also, the small quantities of DNA obtained from
lobsters has declined from over AUS $600 million for 2003–2004 the guts of phyllosomata makes DNA analyses highly susceptible to
to under AUS $400 million for 2009–2011 [2]. exogenous DNA contamination. These issues can be circumvented
Determining the diet of spiny lobster larvae has been difficult, through sequencing a greater number of amplicons, which would
because the low densities and patchy distribution of these animals allow genuine sequences to be identified amongst sequences that
in the open ocean makes them difficult to observe and it is are PCR and laboratory artefacts [36]. Therefore, the present
perceived to be prohibitively expensive to conduct blue water field study employs a high-throughput DNA sequencing approach to
studies [17]. Microscopic analysis of the gut contents of attempt to overcome these shortcomings, and represents a
phyllosomata is also a difficult way to determine diet because significant advance for this methodology for a wide range of such
many potential prey lack hard parts and often have transparent dietary studies. Using these methods over 30,000 amplicons were
body morphology [1]. Researchers have therefore relied on sequenced from the gut contents of eighteen phyllosomata of P.
various methods to infer the diet of phyllosomata of several cygnus (stage VI and VII) taken from a single water mass 150 km
species. These methods include fatty acid profiling of wild offshore of Western Australia in an effort to establish if these
phyllosomata [8,18], captive feeding trials [19,20], examining methods can more accurately determine the composition of the
limb, gut and mouthpart physiology [21–23], enzyme profiling diet of phyllosomata.
[24], stable isotopes [25], and the sequencing of DNA from gut
contents [26–29]. These methods, combined with insights from Methods
the artificial culture of phyllosomata, suggest that these larvae may
be generalist predators that consume gelatinous zooplankton such Ethics Statement
as Chaetognatha (arrow worms), Cnidaria, fish larvae, Salpa and Collection and preparation of the phyllosoma material was
soft-bodied arthropods. However, many details are still uncertain, conducted under Western Australian Department of Fisheries
including which species or taxonomic groups are targeted in the Research Permit number 1724-2010-38 and Murdoch University
wild, and how this changes spatially, temporally and with animal ethics permit number R2338/10.
developmental stage. Of the methods used to date, the DNA-
based approaches have proved to be the most powerful to study Sampling
the diet of phyllosomata because they have the capability to assign Samples were collected from the RV Southern Surveyor (CSIRO,
taxonomy to digesta obtained from the larval gut with a degree of Australia) on 7, 13 and 14 July 2010 (refer Table 1 for geographic
resolution that other methods are not capable of. co-ordinates). Surface waters were sampled at night with a surface
In previous DNA studies PCR has been performed with net (1 m2 opening, 1 mm mesh and cod-end with 355 mm mesh)
universal primers on DNA extracted from the gut contents and a for around 10 minutes at less than 3.7 km hour21. On recovery of
small random selection of PCR amplicons were cloned and the net the contents of the cod-end were poured into shallow
sequenced using traditional techniques. In the first study of its plastic trays. Phyllosomata were immediately sorted out, preserved

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 Larval Lobster Diet

Table 1. Samples. the Chargeswitch ForensicTM DNA extraction kit following the
manufacturer’s instructions. All plasticware used was sterile and
nuclease free. Dissection and DNA extraction were performed in a
Length UV sterilised laminar flow hood following the recommendation of
ID MID MID Date Stage (mm) Latitude Longitude Blankenship and Yayanos [39]. PCR reactions were set up in a
separate UV sterilised PCR hood.
1 MID1 MID9 7-Jul VII 14.2 30.72 113.52
2 MID1 MID10 7-Jul VII 15.5 30.72 113.52
Design of prey enriched PCR
3 MID1 MID11 7-Jul VII 17 30.72 113.52 Phyllosomata potentially prey on a range of zooplankton from
4 MID1 MID13 7-Jul VII 17.1 30.72 113.52 phylogenetically divergent phyla, which restricts potential loci to
5 MID1 MID14 13-Jul VII 14.5 30.70 113.83 those gene regions with highly conserved priming sites. The DNA
6 MID2 MID9 13-Jul VII 16 30.70 113.83 from the digesta removed from the hepatopancreas was also likely
to be degraded into short fragments. Therefore, the hypervariable
7 MID2 MID10 13-Jul VI 13.9 30.70 113.83
v7 and v9 regions of the 18S rRNA were targeted (Table 2). These
8 MID2 MID11 14-Jul VII 17.2 30.21 113.09
are flanked by highly conserved priming sites and diverse
9 MID2 MID14 14-Jul VI 14 30.21 113.09 assemblages of meiofauna and microscopic eukaryotes have been
10 MID3 MID9 14-Jul VII 14.4 30.21 113.09 amplified for v7 [40,41] and v9 [42,43]. Details on two primers
11 MID3 MID10 14-Jul VII 17.2 30.21 113.09 used in this study have been published previously [44,45]. The
12 MID3 MID11 14-Jul VI 10.5 30.21 113.09
other two primers target a similar region to that used in previously
published studies, but have been moved slightly to enable the
13 MID3 MID13 14-Jul VI 14 30.21 113.09
priming of more metazoan phyla, in particular Cnidaria,
14 MID3 MID14 14-Jul VII 17.5 30.21 113.09 Ctenophora and Chaetognatha: 18S_v7_con is a version of
15 MID4 MID9 14-Jul VI 12.5 30.21 113.09 Uni1304F primer of Larsen et al [46] and 18S_v9_con targets a
16 MID4 MID10 14-Jul VI 17.8 30.26 113.05 region slightly 59 (upstream) of the popular NSF1624 primer [47].
17 MID4 MID11 14-Jul VI 14 30.26 113.05
Because these universal primers would otherwise amplify lobster
DNA, they were used in conjunction with a PNA-clamp to
18 MID4 MID13 14-Jul VI 13.5 30.14 113.34
suppress the amplification of lobster DNA.
ext neg MID7 MID9 Prey enrichment has been performed in other DNA diet studies
ext neg MID7 MID10 using DNA blocking primers with 39 termini modified to prevent
PCR neg MID7 MID11 polymerisation [30]. However, the 18S rRNA of some potential
PCR neg MID7 MID13 prey, specifically the arthropods, differs little from that of lobsters
and if a modified DNA primer were used then there might be non-
Stage, length and source loction of phyllosoma larvae used in study as well as specific binding. Therefore, PNA-clamps were used because their
duplicate negative (no template) controls for contamination originating from low mismatch tolerance would reduce non-specific PCR enrich-
DNA extraction (ext. neg) and PCR (PCR neg). Date refers to when the sample
was collected and length refers to the distance from the top of the cephalic
ment [48], thereby building on the use of PNA to ascertain the diet
shield to the bottom of the abdomen. MIDs refer to Roche’s multiplex of larval lobsters by Chow et al. [29]. In contrast to them, this
identifiers that uniquely identify samples in the 454 GS reaction [49]. study did not use the PNA to competitively exclude the binding of
doi:10.1371/journal.pone.0042757.t001 PNA primers to predator template, but rather to bind downstream
of the primers and arrest polymerisation. The benefit of using this
in 70% EtOH and then stored on board the vessel at 220uC for arrest approach is that the enrichment is not constrained to
later analysis in the laboratory. targeting regions of DNA where priming sites were immediately
adjacent to hyper-variable clamping sites, but could independently
DNA extraction target regions that were ideal primer and PNA clamping sites,
Species identity of phyllosomata was confirmed by sequencing which allowed us to design a very reliable PCR [33]. Therefore,
the mitochondrial cytochrome-oxidase I gene (COI). For this, the PNA in this study can be used to prevent and enrich the prey
approximately 1 mm of the fifth pereiopod was removed and of any phyllosomata from the Palinuridae or Scyllaridae.
DNA extracted using the prepGEMTM extraction kit (Zygem,
Hamilton, New Zealand) following manufacturer’s instructions, PCR
but in 20 mL reagent volume. PCR used the LCO-1490 and PCR amplification was undertaken in two rounds following the
HCO-2198 primers and PCR protocol of Folmer et al. [37] except ‘‘universal tailed amplicons sequencing’’ method outlined in the
20 mL reactions were used. Phyllosomata were staged under a GS Junior System Guidelines for Amplicon Sequencing [49]. In
dissecting microscope according to the developmental key of the first round of PCR the hypervariable v7 and v9 regions of the
Braine et al. [38]. 18S rRNA gene were targeted in separate reactions (refer Table 2
In the laboratory, phyllosomata were rinsed with 600 mL of for primer sequences). PCR products were diluted 1:50 and 2.5 ml
sterile MQ water using wash bottles to remove any loosely used as template in a subsequent reaction to add adapter A and B
adhering surface contaminants. Phyllosomata were then mounted sequences, the 454 GS-FLX Titanium sequencing key and
in solidified 2% agar gel so that their cephalic region was exposed. multiplex identifier tags (MIDs). Each PCR was carried out in
Gut content was syringed out using individual, sterile, disposable 25 mL volumes using a GeneAmp 9700 thermocycler (Applied
31 gauge hypodermic needles (Ultra-fine II, Becton Dickinson, Biosystems Foster City, CA, USA). Reactions contained 16
Australia). These were first flushed with ChargeswitchTM DNA reaction buffer, 2 mM of MgSO4 (Invitrogen) 0.46 BSA (NEB),
extraction buffer (Invitrogen, Carlsbad, CA), mounted on a 0.1 mM dNTPs (Roche), 0.1 mM of forward and reverse primers
micromanipulator and then carefully inserted into the hepatopan- (IDT), 1 mM of PNA-clamp (Panagene) and 1 unit of Hi-fidelity
creas of the phyllosomata and care was taken to minimise contact Platinum Taq (Invitrogen). First round PCRs contained 25 ng
with the animal’s exterior. DNA extraction was performed with genomic DNA or were negative (no template) controls for

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 Larval Lobster Diet

Table 2. Locus Specific Primers.

Name Target Sequence Reference

M13F_EukB 18S v9 TGT AAA ACG ACG GCC AGT TGA TCC TTC TGC AGG TTC ACC TAC [44]
M13R_18s_v9_Con 18S v9 CAG GAA ACA GCT ATG ACC CCT TTG TAC ACA CCG CCC This study
M13R 18S_v7_Con 18S v7 CAG GAA ACA GCT ATG ACG CCG TTC TTA GTT GGT GGA This study
M13F All18SR 18S v7 TGT AAA ACG ACG GCC AGT CAT CTA AGG GCA TCA CAG ACC [45]
Lobster_PNA_18S_v7_17mer 18S v7 TTG CGA ACG GAC ACC AC-Lys This study
Lobster_PNA_18S_v9_18mer 18S v9 CGC TCT TGG ATG TTC TAC-Lys This study

Primers used in first round of PCR. Tagged fusion primers used in second round follow Roche guidelines [49] and are available on request.
doi:10.1371/journal.pone.0042757.t002

contamination during DNA extraction or contamination. The first The frequency of different organisms was then returned for each
round of the v7 region reaction was carried out at 94uC for 2 min, of the seven taxonomic levels. This approach, of clustering
followed by 28 cycles of 94uC for 20 s, 56uC for 20 s, and 68uC for sequences based on their best taxonomic hits also mitigates the
12 s followed by a final extension step of 68uC for 30 s. The first problem of grossly overestimating taxon richness that can result
round of the v9 region reaction differed, following a protocol of from directly clustering sequences into OTUs [52].
94uC for 2 min, followed by 30 cycles of 94uC for 20 s, 59uC for To assess if there was sufficient sequencing coverage to capture
20 s, and 68uC for 12 s followed by a final extension step of 68uC prey richness a rarefaction curve was generated for both the v7
for 30 s. Second round PCR reactions used the following protocol: and v9 loci. For this, an OTU approach was determined to be
94uC for 2 min, 6 cycles of 94uC for 20 s, 58uC for 20 s, and 68uC preferential because resolving taxonomy to fine levels is not reliant
for 12 s, followed by 14 cycles of shuttle PCR of 94uC for 20 s, on the completeness of the reference database. OTUs were
then 68uC for 30 s and a final extension step of 68uC for 30 s. generated at 93%, 95% and 97% similarity thresholds and curves
PCR amplicons, which contained 454 GS-FLX Titanium fusion were calculated with the software Analytic Rarefaction 2.0 [53]
primers and MID sequences, were separately cleaned using that implements the equations of Tipper [54].
Ampure XPTM beads (Agencourt) following the manufacturer’s
instructions. Ampure XPTM beads were calibrated according to Results
Roche 454 GS Junior Methods Manual [50] and amplicons over
200 bp were size selected. Amplicons were run on the Agilent PCR amplicon sequences from the hepatopancreas of
Bioanalyzer (Agilent Technologies, Germany GmbH) with DNA phyllosomata
1000TM chips to check the quality and size distribution of A total of 100,693 sequencing reads were returned by the 454
amplicons. Amplicons were then diluted, pooled, re-cleaned with GS Junior. Out of these reads 36,800 passed the stringent quality
Ampure XPTM and triplicate samples were quantified the using control requirements that both MIDs and primers had no
Qubit Fluorometer (Invitrogen) and quality control repeated on mismatched bases. The average number of reads for each sample
the Agilent Bioanalyzer. After quality control, the pooled was 2041, and of these, the average number for the v7 locus was
amplicons were diluted to 16109 molecules ml21 ready for 752 (n = 18) and 1289 for the v9 (n = 18).
sequencing following the Roche 454 GS Junior workflow.
Taxonomic identity of DNA from samples
Bioinformatics Of the quality controlled reads, 8,628 were a close match to
Basic bioinformatics were performed using custom PERL scripts lobster, with 3,837 reads coming from the v7 loci and 4,791 reads
and a brief summary follows. Amplicon reads were assorted to coming from v9. Although numerous lobster reads occurred in the
individual samples based on 454 MIDs and then split into the v7 dataset, none of these reads contained the PNA-clamp sequence.
and v9 18S rRNA loci using the PCR primer sequence. To quality The negative controls for PCR reactions were empty for v9, but v7
control the sequencing output, any reads that did not perfectly yielded a small number of reads from vascular plants (probably
match the MID or primer sequences were discarded. Reads were pollen) and mammals (probably humans). The negative controls
dereplicated, trimmed and then BLASTed [51] against P. cygnus. A for DNA extractions yielded more DNA in the v7 and v9 loci,
script was subsequently used to BLAST reads that did not match these were also from mammals, vascular plants (v9) and fungi. The
P. cygnus to the NCBI nucleotide database as at August 2011. The negative control for the extraction also contained nine unidentified
ten top-scoring BLAST hits were then returned, or in the case sequences and seven sequences for Bacillariophyta. As none of
where the top BLAST hits were unannotated environmental these taxa are relevant to this study and are most likely sampling or
sequences, the top one hundred hits were returned. Due to the laboratory contaminants, the negative controls were excluded
nature of the BLAST (multiple good hits for a given query from the study, as were any occurrences of the taxa identified in
sequence), a method was adopted that summarised the taxonomic the negative controls if they occurred in other samples.
classification by checking for consistency amongst the top 10 hits. Samples 1, 2, 3, 5 and 11 yielded almost no zooplankton
In the cases where the most closely-related sequence could not be sequences and consequently a proportionately higher percentage
easily determined, it was required that 6 out of the 10 hits of contamination, fungus or Palinuridae (i.e. predator) sequence
belonged to the same taxon otherwise the taxonomic identity was (Fig. 1). This is consistent with there being no metazoan prey tissue
labelled as ‘‘No consensus’’. This was summarised at each of the in the hepatopancreas of the larvae and the PCR reaction
seven taxonomic levels (Kingdom, Phylum, Class, Order, Family, amplifying any available template. These samples were excluded
Genus and Species). In the cases where there was a single best from further analysis. Sample 7 was negative for the v7 loci but
BLAST hit, then this was used as the final taxonomic classification. yielded ample prey-reads in the v9 region. The sequencing results

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 Larval Lobster Diet

for sample 7 in the v7 region were similar to those for the v7 database for v9 we were unable to resolve any higher order
negative controls (i.e., contained reads for mammals and fungus); taxonomic assignments with this higher level of information.
this suggests that there was insufficient template in the initial v7
PCR reaction. Accordingly, sample 7 was only included in Discussion
subsequent analysis using the v9 region and where the v7 and v9
regions were compared it was excluded. Pyrosequencing
Once non-prey reads had been determined and removed from The present study indicates that high throughput sequencing of
samples the average number of prey reads for the v7 locus was short 18S rDNA PCR amplicons is a robust solution to the
546.7 (n = 12, range: 62–1280) and 1103.1 reads for the v9 locus problem experienced in previous DNA diet studies of phylloso-
(n = 12, range: 34–2805). The slopes of the saturation curves mata where the prevalence of non-target amplicons inhibited
rapidly approached asymptotes, which indicates that although detection of prey amplicons [26,28,29]. High throughput
there are multiple read types, sufficient sequencing reads were sequencing technologies have had an immense impact on studying
generated to capture major prey items and trends towards bacterial community composition through mass sequencing of the
capturing full taxon richness (Fig. 2). Rank abundance of DNA 16S rDNA (e.g. [55]) and have gradually become significant for
studies of complex eukaryotic assemblages [56–59]. Compared to
reads showed that five taxa were very highly represented in both
other high throughput sequencing technology the Roche 454
loci, that less than ten taxa were highly represented overall, and
genome sequencer (454 GS-FLX) enables relatively long read
the remainder were represented in very small numbers (Fig. 3).
length [60]. While other sequencing technologies are increasing
The most abundant reads in both loci, were from the phyla and
their read length, the substantial lengths achieved with the 454
classes: Radiolaria (Polycistinea), Chordata (Thaliacea), Chordata
GS-FLX have made this platform an attractive approach for ‘‘bar-
(Actinopterygii), Cnidaria (Hydrozoa) and Chaetognatha (Sagit-
coding’’ based ecological studies including diet studies [31,61,62].
toidea), with these five taxa together composing 97.1% of v7 reads
The 454 GS has been re-released in a smaller scale ‘‘Junior’’
and 93.2% of v9 reads. In addition to these top-ranking classes, platform that is affordable for a moderate-sized laboratory (Roche
Malacostraca and Gastropoda contributed about 2% and 0.5% to 2011). Using this affordable technology the present study was able
each locus respectively. to exclude contaminants and chimeras during bioinformatic
There were minor differences in the results between the v7 and analyses without having a dramatic impact on the quantity of
v9 loci with slightly more taxa uncovered using the v9 locus than sequence reads for analysis. This would not be possible with the
the v7 locus (Table 3), although, these taxa were generally number of sequence reads afforded in a traditional cloning effort.
represented by less than 1% of reads. Ascidiacea (Chordata) By targeting the 18S ribosomal gene the present study overcame
occurred exclusively in the v9 locus. Discrepancies are most likely the problems that Chow et al. (2010) identified using the ultra-
due to PCR amplification biases from primer binding, but these variable ITS1 region, that is, being unable to find sufficiently
biases are small and do not impact on the detection of significant homologous sequences and the problem of length variation
taxa, or even mildly significant taxa, because there is sufficient influencing PCR efficiency. However, a disadvantage of targeting
coverage to ascertain the range of taxa in a sample, which is short 18S regions is that the reads generally contain only sufficient
evidenced by the species accumulation plot (Fig. 2). The v9 region information to determine their taxonomy to either the order or
is more variable than the v7. Therefore, although the same orders family level. This disadvantage was a deliberate trade-off
of taxa were identified with these loci when a clustering approach considered in our experimental design, but it was more important
is used the v9 assigns reads into more OTUs at similar similarity for our immediate questions that the PCR primers were capable of
thresholds (Fig 2). Unfortunately, because of gaps in the reference amplifying all metazoans, which is amply demonstrated by the

Figure 1. Source of sequence reads among individual larvae. Relative distribution of combined reads for each sample to ascertain the
proportion of potential prey reads against other kinds of reads. Samples 1, 2, 3, 5 and 11 had very little potential prey DNA, whereas samples 4, 7, 8,
10, 12, 13, 15 and 17 contained over 50%. Fungal DNA could originate from laboratory contamination, but along with algae, it is just as likely to
originate from the gut and is not relevant to the current study. Contamination was either mammalian (probably human) or plant material and
sequencing artefacts were mostly very short reads.
doi:10.1371/journal.pone.0042757.g001

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 Larval Lobster Diet

Figure 2. Sampling saturation of (a) v7 and (b) v9 loci. Rarefaction curves representing the number of OTUs detected in pooled samples for
the (a) v7 and (b) v9 loci. OTUs are defined at 93%, 95% and 97% respectively and as the percentage threshold increases so does the number of OTUs
detected. However, the estimate of OTU richness for each OTU threshold tends toward an asymptote indicating that there is sufficient sequencing
coverage to detect most taxa.
doi:10.1371/journal.pone.0042757.g002

wide range of taxa revealed from the digesta of the phyllosomata. greater biological significance. However, the numbers of each
Few loci besides the 18S rRNA gene have high coverage in public sequence type can be influenced by a multitude of factors beyond
DNA sequence databases and also have primer-binding sites that prey concentration in the gut. Multi-copy genes such as ribosomal
are conserved across the Cnidaria, Ctenophora, Chaetognatha, genes vary in copy number across different animals [63]. Read
Urochordata and Vertebrata. An unexpected benefit of choosing number may also vary due to PCR amplification bias prior to
such conserved loci was the discovery of a predominance of DNA sequencing [64,65]. Starting with a low quantity of genomic DNA
from colonial radiolaria, which was otherwise unanticipated and template can also cause stochastic sampling errors to distort
has rarely, if ever, been considered as a potential prey for amplicon composition [66,67]. To a limited extent amplification
phyllosomata [1]. Another advantage of the conserved nature of bias was anticipated and controlled by targeting two loci on the
the 18S rRNA is that the PNA-clamps work with all lobster species same gene. If there were little amplification bias it would be
from the families Palinuridae and Scyllaridae. This is particularly predicted that the same taxa could dominate rank abundance for
attractive because this protocol and its reagents can be reused to v7 and v9 (Fig. 3) and that taxa would be ranked in the same
study the structure of the diets of the larvae of other species of order. The concordance between the results for these two loci
lobster, which makes the approach very economical. indicates that PCR bias had little negative impact on determining
There was DNA for multiple taxa in the hepatopancreas of each prey items, and therefore relative abundance of reads at the level
phyllosoma, with the exception of phyllosoma 7, which returned of class and order may reflect some relative differences in the
339 reads that exclusively matched chaetognatha, indicating consumption of prey species for each phyllosoma. This inference
Chaetognatha tissue as its sole dietary source. The temptation on the relative abundance of reads also tends to be supported by
with community amplicon sequencing is to treat the sequence the overall general concordance of these data with the dietary
reads as quantitative or semi-quantitative, and therefore to treat profile among multiple phyllosomata provided by the presence/
higher frequency operational taxonomic units (OTUs) as having absence of DNA for the same range of taxa (Table 3).

Figure 3. Rank abundance of potential prey reads after standardisation. Rank abundances for the (a) v7 and (b) v9 loci. The x-axis shows the
prey classes that constituted more than 0.5% of the sequencing reads. Samples were standardised into ratios of prey per predator prior to combining
them into rank abundance.
doi:10.1371/journal.pone.0042757.g003

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 Larval Lobster Diet

Table 3. Presence/absence of prey items across samples. analysis and comparable to the price of lipid analyses that have
much less power for taxonomic resolution [8,18]. Therefore, the
better resolution of an enriched meta-genetic approach makes it a
v7 18S rDNA valuable tool for trophic analyses of phyllosomata and other
v9 18S rDNA
pelagic predators.
Class Order Frequency (n = 12)

Malacostraca Amphipoda - 2 Larval diet: taxa from gut DNA

Decapoda 2 (Pandalidae) 2 (Pandalidae) The dataset from this study indicates that gelatinous zooplank-
Euphausiacea 8 (Euphausiidae) 8 (Euphausiidae) ton are significant in the trophic ecology of P. cygnus. This is
entirely consistent with the hypothesised diet items that have been
Maxillopoda Calanoida 1 3
inferred from examining the mouthparts and feeding structures of
Calanoida 1 (Metridinidae) phyllosomata [1]. Tunicates, fish larvae, siphonophores and
Sagittoidea Aphragmophora 8 (Sagittidae) 8 chaetognaths have been indicated as prey in the DNA diet studies
Actinopterygii No Consensus 9 7 of P. japonicus [26,28,29] as well as in experimental feeding of P.
Thaliacea Doliolida 2 (Doliolidae) 1 interruptus [19].
Pyrosomata 2 (Pyrosomatidae) -
The discovery of DNA from colonial radiolarians in the guts of
phyllosomata is a new addition to the groups of organisms
Salpida 9 (Salpidae) -
associated with the diet of phyllosomata. Colonial radiolarians fit
No Consensus - 10 the profile of potential prey for middle to late stage phyllosomata
Hydrozoa Hydroida 1 1 because they are gelatinous zooplankton that cannot easily evade
No Consensus 2 6 capture [1]. The zooplankton assemblages in East Indian Ocean
Siphonophora 7 6
eddies have typically been assessed using plankton nets, a sampling
technique that tends to destroy delicate zooplankton, and probably
Trachylina 9 4
greatly under-represents colonial radiolarian density [69]. Less
Scyphozoa No consensus - 1 destructive sampling techniques such as video plankton recorders
Tentaculata No consensus - 3 (VPR) demonstrate that net tows of identical water bodies under-
Holothuroidea Apodida 1 2 represent the true concentration of colonial radiolarians by at least
Cephalopoda Teuthida 3 2 an order of magnitude and often by more than several orders [69].
The only video plankton recorder study of East Indian Ocean
Gastropoda Thecosomata 2 5
oceanic waters offshore from the coast of Western Australia found
No Consensus - 2 that colonial radiolarians were by far the most dominant member
Polycystinea Spumellaria 12 10 of the zooplankton assemblage [70]. This abundance is not
surprising because colonial radiolarians are very well suited to the
The frequency of prey taxa occurring across the twelve phyllosomata that
contained traces of prey in the gut. Prey were identified to the hierarchical level
extreme oligotrophic conditions found in this oceanographic
of order and brackets denote the family-level of assignment to a sequence region. The colonial radiolarian DNA identified in this study
where it could be determined. ‘‘No consensus’’ refers to taxa that have too little came from the families Sphaerozoidae and Collosphaeridae,
database coverage to confidently assign a taxonomic order to the sequences. which are monophyletic clades that are exclusively colonial and
doi:10.1371/journal.pone.0042757.t003
lack skeletons [71]. The Sphaerozoidae has species whose colonies
can reach dimensions of over 2–3 m [72,73] making them large
This study targeted very short reads of around 150 bp (v7) and targets for passive encounter by drifting phyllosomata in the water
180 bp (v9). Although larger DNA fragments are useful for column, and they could serve as both a refuge and food source for
taxonomic assignment, they are in low concentrations in digesta phyllosoma.
[68] and have proven difficult to amplify in diet studies of
phyllosomata [26]. Using very little DNA template and 50 PCR Interpreting multiple reads from hepatopancreas
cycles this study generated sufficient amplicons to profile DNA Recovering amplicons from multiple taxa in each phyllosoma
from the hepatopancreas of the larvae, which is an achievement sample was not unprecedented. In their cloning based study Chow
made possible by targeting shorter reads. Results from this study et al. [29] detected 0–4 taxa for each phyllosoma and one
indicate that there are a sufficient number of amplicons generated phyllosoma contained 10 different prey types. Of these reads,
through PCR, suggesting that fewer PCR cycles could be used in thirteen were from Ctenophora, six and four were unidentified
future work, which would be advantageous because it would homologues and the other seven reads were singletons. The
reduce the accumulation of PCR artefacts. This would not only presence of multiple reads could have several explanations; some
reduce the number of base miscalls in prey DNA, but also reduce reads are possibly sampling artefacts, phyllosomata may prey on
the number of lobster amplicons that have incorporated a multiple taxa, some taxa may be from secondary predation, and
replication error in the PNA-clamp region, which enables them phyllosomata may engage in coprophagy. These possibilities are
to elude enrichment. It might also reduce the discrepancies discussed in detail below.
between the v7 and v9 loci. Sampling artefacts could result from phyllosomata opportunis-
The approach presented in this study not only builds on DNA- tically feeding on animals in the cod-end of the zooplankton net or
based approaches to study the diet of phyllosomata, but it is a subsequently in sorting trays. Opportunistic net feeding has been
promising alternative to other methods. The cost of this method reported in ecology studies for other predators [74], however, this
was around US $200 per locus per sample. However, because this seems unlikely as phyllosomata appear to become incapacitated
value includes the initial optimisation of reactions, the purchase of once captured in nets due to the fragility of their feeding
bulk reagents, and due to the ever-decreasing cost of sequencing, it appendages. Phyllosomata of slipper lobsters (of the same infra-
is anticipated that this cost will drop to less than US $100 per locus order as P. cygnus) have been observed in the wild to cling to
per sample. This price is more expensive than stable isotope Cnidaria [75,76,77]. The Cnidaria could be prey, transport or

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 Larval Lobster Diet

both. This close association between the animals means that there in these conditions, so if the present study detected DNA from
is a risk that exogenous DNA adhering to the predator’s exterior faecal pellets then they would have been recently excreted. For
could be detected through PCR and subsequent sequencing. To example, faecal pellets from tunicates are rapidly colonised and
minimise this risk the exterior of the animals were washed when assimilated by microbes and other zooplankton that could rapidly
collected and prior to dissection, also the gut contents were digest residual DNA [90]. If it occurs, then coprophagy is likely to
syringed out of the hepatopancreas through a small gauge needle. be an opportunistic feeding strategy by phyllosomata and not their
By taking these measures the overwhelming majority of DNA primary dietary source because captive feeding studies and the
extracted for this study came from the predator’s interior. The physiology of phyllosomata all demonstrate active predation of
presence of multiple taxa inside each phyllosoma may be zooplankton [20,78]. However, it is logical that a passive
explained by a recent history of feeding on several different prey encounter feeder in oligotrophic waters will opportunistically
items. Examination of phyllosomata in culture conditions has consume marine snow. So despite the strong evidence that
demonstrated that they can be voracious feeders that are capable phyllosomata are active predators, marine snow and faecal pellets
of rapidly processing prey [20,78,79]. The digestive gland may potentially act as dietary subsidies. Eel larvae, or leptocephali,
structure also includes a series of blind diverticula within which which like phyllosomata are a common feature of oligotrophic
digestion processes may progress relatively slowly in comparison to ocean waters, appear to have specialised in feeding on marine
the rate at which prey may be consumed [78]. Therefore, it is snow, especially the faecal pellets of zooplankton and discarded
conceivable that the gut contents of phyllosomata are a larvacean houses [91,92].
representation of recent prey feeding history in terms of the
residual DNA retained throughout the extent of the digestive Conclusion. Phyllsoma Diet and the Western Australian
gland. Of the eighteen phyllosomata examined, five had no prey Fishery
DNA signal, possibly due to recent absence of prey encounter, or a The variety of gelatinous zooplankton that this study detected,
temporary halt to feeding which is known to occur when and the possibility of a faecal subsidy, support the hypothesis that
phyllosomata enter a moulting event [80]. Of the remaining phyllosomata are generalist, opportunistic feeders of this type of
thirteen phyllosomata, colonial radiolarian DNA was predominant prey. However, while this opportunism might provide a basal
and was detected in twelve phyllosomata suggesting that it is a level of sustenance, it may be insufficient to deliver excess energy
commonly consumed prey species. Thaliacea, predominantly to store as lipids that are needed to fuel the migration of the
Salpa, were the next most abundant class and were present in pueruli to shore. Further work is required to ascertain the optimal
ten phyllosomata. Actinopterygii, Hydrozoa and Sagittoidea were food for phyllosomata and whether oceanographic processes
each detected in nine larvae (Fig 3). impact the density of optimal prey groups and/or the ability of
DNA dietary studies can be very sensitive to secondary phyllosomata to capture these prey. Attempts to culture
predation, where the food species that remains present in the phyllosomata have found that feed is a strong determinate of
digestive tract of the prey is co-amplified with prey DNA [81]. larval viability, and supplementation of feed with mussel gonad
Phyllosomata can rapidly consume their entire prey [1], so it is has been found to be essential for phyllosomata to advance
likely that phyllosomata are also consuming the gut content of through multiple instars (Kittaka 1997). More recently, a study of
their prey. Directly targeting the gut of zooplankton is not an wild-caught mid-late stage P. cygnus phyllosomata reared under
unusual trophic strategy in oligotrophic water [82], as the gut culture conditions and offered live freshly caught chaetognatha,
contains a conveniently concentrated and partially digested source salpa or krill, showed a clear preference for, and an ability to
of plankton. The gut/pseudogut contents of Urochordata, consume significant numbers of chaetognatha [20]. It was also
Cnidaria and Ctenophora are limited only by mouth size and shown that phyllosomata experimentally fed on chaetognatha
many of the low abundance amplicons detected, such as over six days tended to accumulate lipid, which is known to be
Bacillariophyta, could well have been the food species of prey. important for energy storage in phyllosomata for subsequent use
Likewise, colonial radiolarian are known to consume various during the pueruli migration to the coast [8,13,20]. The spatial
zooplankton upon making contact with them [83]. Although and temporal variability in the availability of these significant
Chaetognatha are most often described as preying on copepods, prey items could result in phyllosomata in poor nutritional
this is most likely because most studies on chaetognatha have taken condition, which in turn may contribute to pueruli having
place in eutrophic waters where they are often an abundant and insufficient lipid reserves to recruit back to the shallow coastal
important predator of grazing copepods [84]. However, the environment [8,14]. Together, the results of these studies indicate
Chaetognatha have been found to consume various other prey that if the most nutritionally significant prey items of phylloso-
such as Tinnitids, Appendicularia [85] and Euphausiacea [86]. mata can be determined, it would allow for the monitoring and
Chaetognatha also digest only around 80% of their prey [87], modelling of how oceanic dynamics impact on these prey that
which would provide sufficient residual DNA for it to be detected would improve in the understanding of recruitment and assist in
as secondary predation in our study. managing the rock lobster fishery.
Many zooplankton opportunistically consume detritus (marine Without knowing the diet of phyllosomata it is not possible to
snow) such as faecal pellets, discarded appendicularian ‘houses’ test the hypothesis that spiny lobster larval recruitment depends on
and zooplankton carcasses [88,89]. In captive feeding studies P. successful feeding of the oceanic phyllosomata to fuel the
cygnus were observed to frequently capture small pieces of shoreward migration of non-feeding pueruli. The DNA approach
suspended detritus to feed on (pers ob). If phyllosomata consumed presented in the present study identifies a group of key animals
detritus it would provide a further explanation for the detection of that are clearly important in the trophic ecology of P. cygnus
multiple taxa taken from gut samples. If phyllosomata are phyllosomata. While this approach cannot unequivocally establish
coprophagic then the most abundant DNA reads from the that an animal is directly preyed upon, it is highly informative for
hepatopancreas are likely to be from the animal that excreted providing an indication of the potential nutritional importance of
the particle and the sequences in lower abundance would be traces prey taxa. For example, the results indicate colonial radiolarians
of that animal’s prey. However, because DNA is an excellent are a predominant constituent of the prey DNA of phyllosomata.
source of carbon, phosphate and nitrogen it is unlikely to last long Although it cannot be confirmed that phyllosomata feed directly

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 Larval Lobster Diet

on colonial radiolarians, or via coprophagy or secondary Acknowledgments

predation, such as through chaetognatha, it remains the first time
We are grateful to the Captain, crew and scientific team of R. V. Southern
to our knowledge that colonial radiolarian have been associated
Surveyor voyage 2010-05. Liam Williams of the University of Auckland
with the diet of phyllosomata. Other taxa for which DNA was CGP (now a part of NZGL) for his knowledgeable input into the
commonly found in phyllosomata, included salpa, fish larvae, sequencing process. Nick Caputi and Fisheries WA for their support and
siphonophora and chaetognatha suggesting they are also likely to advice.
be significant to the trophic ecology of P. cygnus. The high-
throughput amplicon sequencing approach presented in the Author Contributions
present study enables further discovery of how the trophic Conceived and designed the experiments: RO SL AJ SC HT. Performed
connections of P. cygnus are spatially and temporally structured the experiments: RO. Analyzed the data: RO PT. Contributed reagents/
and how this relates to the nutritional condition of the materials/analysis tools: SL LB AW AJ. Wrote the paper: RO SL AJ.
phyllosomata. Sample collection: LB PAT AW.

References
1. Jeffs AG (2007) Revealing the natural diet of the phyllosoma larvae of spiny western rock lobster (Panulirus cygnus, Decapoda: Palinuridae). J Morph 220: 271–
lobster. Bull Fish Res Agen 20: 9–13. 280.
2. ABARES (2011) Australian fisheries statistics 2010. Canberra. 109 p. 23. Cox S, Johnston D (2004) Developmental changes in foregut functioning of
3. Phillips B, Brown P, Rimmer D, Reid D (1979) Distribution and Dispersal of the packhorse lobster, Jasus (Sagmariasus) verreauxi (Decapoda: Palinuridae), phyllo-
Phyllosoma Larvae of the Western rock Lobster, Panulirus cygnus, in the South- soma larvae. Mar Freshwater Res 55: 145–153.
eastern Indian Ocean. Aust J Mar Freshwater Res 30: 773–783. 24. Johnston D, Ritar A, Thomas C (2004) Digestive enzyme profiles reveal digestive
4. Phillips BF, McWilliam PS (2009) Spiny lobster development: where does capacity and potential energy sources in fed and starved spiny lobster (Jasus
successful metamorphosis to the puerulus occur?: a review. Rev Fish Biol edwardsii) phyllosoma larvae. Comp Biochem Physiol B 138: 137–144.
Fisheries 19: 193–215. 25. Waite A, Muhling B, Holl C, Beckley L, Montoya J, et al. (2007) Food web
5. Caputi N, Chubb C, Pearce A (2001) Environmental effects on recruitment of structure in two counter-rotating eddies based on d15N and d13C isotopic
the western rock lobster, Panulirus cygnus. Mar Freshwater Res 52: 1167–1174. analyses. Deep Sea Res II 54: 1055–1075.
6. Pearce A, Phillips B (1988) ENSO events, the Leeuwin Current, and larval 26. Suzuki N, Murakami K, Takeyama H, Chow S (2006) Molecular attempt to
recruitment of the western rock lobster. J Cons int Explor Mer 45: 13–21. identify prey organisms of lobster phyllosoma larvae. Fish Sci 72: 342–349.
7. Caputi N (2008) Impact of the Leeuwin Current on the spatial distribution of the
27. Suzuki N, Murakami K, Takeyama H, Chow S (2007) Eukaryotes from the
puerulus settlement of the western rock lobster (Panulirus cygnus) and implications
hepatopancreas of lobster phyllosoma larvae. Bull Fish Res Agen 20: 1–7.
for the fishery of Western Australia. Fisheries Oceanogr 17: 147–152.
28. Suzuki N, Hoshino K, Murakami K, Takeyama H, Chow S (2008) Molecular
8. Phillips B, Jeffs AG, Melville-Smith R, Chubb C, Nelson M, et al. (2006)
Diet Analysis of Phyllosoma Larvae of the Japanese Spiny Lobster Panulirus
Changes in lipid and fatty acid composition of late larval and puerulus stages of
japonicus (Decapoda: Crustacea). Mar Biotechnol 10: 49–55.
the spiny lobster (Panulirus cygnus) across the continental shelf of Western
Australia. Comp Biochem Physiol B 143: 219–228. 29. Chow S, Suzuki S, Matsunaga T, Lavery S, Jeffs AG, et al. (2010) Investigation
9. Limbourn AJ, Babcock RC, Johnston DJ, Nichols PD, Knott B (2009) Spatial on Natural Diets of Larval Marine Animals Using Peptide Nucleic Acid-
and temporal variation in lipid and fatty acid profiles of western rock lobster Directed Polymerase Chain Reaction Clamping. Mar Biotechnol 13: 305–313.
pueruli at first settlement: biochemical indicators of diet and nutritional status. 30. Vestheim H, Jarman S (2008) Blocking primers to enhance PCR amplification of
Mar Freshwater Res 60: 810–823. rare sequences in mixed samples – a case study on prey DNA in Antarctic krill
10. Limbourn AJ, Nichols PD, (null) (2009) Lipid, fatty acid and protein content of stomachs. Front Zool 5: 12.
late larval to early juvenile stages of the western rock lobster, Panulirus cygnus. 31. Deagle BE, Kirkwood R, Jarman SN (2009) Analysis of Australian fur seal diet
Comp Biochem Physiol B 152: 292–298. by pyrosequencing prey DNA in faeces. Mol Ecol 18: 2022–2038.
11. Jeffs AG (2001) Can compromised condition explain early mortalities in spiny 32. Vestheim H, Deagle BE, Jarman SN (2011) Application of blocking
lobster culture? In: Evans LH, Jones JB, editors. Proceedings International oligonucleotides to improve signal-to-noise ratio. PCR. Methods Mol Biol
Symposium on Lobster Health Management. Perth: Curtin University Press. pp 687: 265–274.
64–74. 33. O’Rorke R, Lavery S, Jeffs AG (2012) PCR enrichment techniques to identify
12. Phleger CF, Nelson MM, Mooney BD, Nichols PD, Ritar AJ, et al. (2001) Lipids the diet of predators. Mol Ecol Res 12: 5–17.
and nutrition of the southern rock lobster, Jasus edwardsii, from hatch to puerulus. 34. Beja-Pereira A, Oliveira R, Alves PC, Schwartz MK, Luikart G (2009)
Mar Freshwater Res 52: 1475–1486. Advancing ecological understandings through technological transformations in
13. Jeffs AG, Chiswell S, Booth J (2001) Distribution and condition of pueruli of the noninvasive genetics. Mol Ecol Res 9: 1279–1301.
spiny lobster Jasus edwardsii offshore from north-east New Zealand. Mar 35. King R, Read D, Traugott M, Symondson W (2008) Molecular analysis of
Freshwater Res 52: 1211–1216. predation: a review of best practice for DNA-based approaches. Mol Ecol 17:
14. Wilkin J, Jeffs AG (2011) Energetics of swimming to shore in the puerulus stage 947–963.
of a spiny lobster: Can a postlarval lobster afford the cost of crossing the 36. Pompanon F, Deagle BE, Symondson WOC, Brown DS, Jarman SN, et al.
continental shelf? Limnol Oceanogr: Fluids Environ 1: 163–175. (2012) Who is eating what: diet assessment using next generation sequencing.
15. McWilliam P, Phillips B (2007) Spiny lobster development: mechanisms Mol Ecol 21: 1931–1950.
inducing metamorphosis to the puerulus: a review. Rev Fish Biol Fisheries 17: 37. Folmer O, Black M, Hoeh W, Lutz R, Vrijenhoek R (1994) DNA primers for
615–632. amplification of mitochondrial cytochrome c oxidase subunit I from diverse
16. Brown RS (2009) Western rock lobster low puerulus settlement risk assessment: metazoan invertebrates. Mol Mar Biol Biotechnol 3: 294–299.
draft report for public comment. Western Australian Department of Fisheries: 38. Braine SJ, Rimmer DW, Phillips BF (1979) An illustrated key and notes on the
1–52.
phyllosoma stages of the western rock lobster Panulirus cygnus. CSIRO Australian
17. Ritz D (1972) Factors affecting the distribution of rock-lobster larvae (Panulirus
Division of Fisheries and Oceanography Perth Australia: 1–10.
longipes cygnus), with reference to variability of plankton-net catches. Mar Biol 13:
39. Blankenship L, Yayanos A (2005) Universal primers and PCR of gut contents to
309–317.
study marine invertebrate diets. Mol Ecol 14: 891–899.
18. Jeffs AG, Nichols P, Mooney B, Phillips K, Phleger C (2004) Identifying
potential prey of the pelagic larvae of the spiny lobster Jasus edwardsii using 40. Chariton AA, Court LN, Hartley DM, Colloff MJ, Hardy CM (2010) Ecological
signature lipids. Comp Biochem Physiol B 137: 487–507. assessment of estuarine sediments by pyrosequencing eukaryotic ribosomal
19. Mitchell J (1971) Food preferences, feeding mechanisms, and related behavior in DNA. Front Ecol Environ 8: 233–238.
phyllosoma larvae of the California spiny lobster, Panulirus interruptus (Randall). 41. Gast R, Dennett M, Caron D (2004) Characterization of protistan assemblages
San Diego State College: 110. in the Ross Sea, Antarctica, by denaturing gradient gel electrophoresis. Appl
20. Saunders M, Thompson P, Jeffs AG, Säwström C, Sachlikidis N, et al. (2012) Environ Microb 70: 2028–2037.
Fussy feeders: Phyllosoma larvae of the western rock lobster (Panulirus cygnus) 42. Pawlowski J, Christen R, Lecroq B, Bachar D, Shahbazkia HR, et al. (2011)
demonstrate strong prey preference. PLoS ONE 7: e36580. doi:10.1371/ Eukaryotic Richness in the Abyss: Insights from Pyrotag Sequencing. PLoS
journal.pone.0036580.g006. ONE 6: e18169.
21. Nishida S, Quigley B, Booth J, Nemoto T, Kittaka J (1990) Comparative 43. Stoeck T, Behnke A, Christen R, Amaral-Zettler L, Rodriguez-Mora MJ, et al.
morphology of the mouthparts and foregut of the final-stage phyllosoma, (2009) Massively parallel tag sequencing reveals the complexity of anaerobic
puerulus, and postpuerulus of the rock lobster Jasus edwardsii (Decapoda: marine protistan communities. BMC biology 7: 72–92.
Palinuridae). J Crust Biol 10: 293–305. 44. Medlin L, Elwood H, Stickel S, Sogin M (1988) The characterization of
22. Lemmens J, Knott B (1994) Morphological changes in external and internal enzymatically amplified eukaryotic 16S-Iike rRNA-coding regions. Gene 71:
feeding structures during the transition phyllosoma-puerulus-juvenile in the 491–499.

PLOS ONE | www.plosone.org 9 August 2012 | Volume 7 | Issue 8 | e42757

 Larval Lobster Diet

45. Hardy CM, Krull ES, Hartley DM, Oliver RL (2010) Carbon source accounting 68. Deagle B, Eveson J, Jarman S (2006) Quantification of damage in DNA
for fish using combined DNA and stable isotope analyses in a regulated lowland recovered from highly degraded samples – a case study on DNA in faeces. Front
river weir pool. Mol Ecol 19: 197–212. Zool 3: 1–11.
46. Larsen J, Frischer M, Rasmussen L, Hansen B (2005) Single-step nested 69. Dennett MR, Caron DA, Michaels AF, Gallager SM, Davis CS (2002) Video
multiplex PCR to differentiate between various bivalve larvae. Mar Biol 146: plankton recorder reveals high abundances of colonial Radiolaria in surface
1119–1129. waters of the central North Pacific. J Plank Res 24: 797–805.
47. Van der Auwera G, Chapelle S, de Wachter R (1994) Structure of the large 70. Stemmann L, Youngbluth M, Robert K, Hosia A, Picheral M, et al. (2008)
ribosomal subunit RNA of Phytophthora megasperma, and phylogeny of the Global zoogeography of fragile macrozooplankton in the upper 100–1000 m
oomycetes. FEBS Lett 338: 133–136. inferred from the underwater video profiler. ICES J Mar Science 65: 433–442.
48. Ørum H, Nielsen P, Egholm M, Berg R, Buchardt O, et al. (1993) Single base 71. Amaral-Zettler L, Anderson O (1999) Towards a molecular phylogeny of
pair mutation analysis by PNA directed PCR clamping. Nucleic Acids Res 21: colonial spumellarian radiolaria. Mar Micropaleontol 36: 67–79.
5332–5336. 72. Swanberg N, Anderson O (1981) Collozoum caudatum sp. nov.: A giant colonial
49. Roche (2010) GS Junior System; Guidelines for Amplicon sequencing. Branford: radiolarian from equatorial and Gulf Stream waters. Deep Sea Res I 28: 1033–
454 Life Sciences Corp. 45 p. 1047.
50. Roche (2011) Technical Bulletin GS FLX System; Short Fragment Removal 73. Swanberg N, Harbison G (1980) The ecology of Collozoum longiforme, sp. nov., a
Procedure. Branford: 454 Life Sciences Corp. 8 p. new colonial radiolarian from the equatorial Atlantic Ocean. Deep Sea Res I 27:
51. Altschul SF, Gish W, Miller W, Myers EW, Lipman DJ (1990) Basic local 715–732.
alignment search tool. J Mol Biol 215: 403–410. 74. Harwood JD (2008) Are Sweep Net Sampling and Pitfall Trapping Compatible
52. Kunin V, Engelbrektson A, Ochman H, Hugenholtz P (2010) Wrinkles in the with Molecular Analysis of Predation? Environ Entomol 37: 990–995.
rare biosphere: pyrosequencing errors can lead to artificial inflation of diversity 75. Thomas L (1963) Phyllosoma larvae associated with medusae. Nature 198: 208.
estimates. Environ Microb 12: 118–123. 76. Shojima Y (1963) Scyllarid phyllosomas’ habit of accompanying jellyfish. Bull
53. Holland SM (2009) Analytic Rarefaction 2.0. Hunt Mountain Software. Jpn Soc Fish 29: 349–253.
Available: www.huntmountainsoftware.com. Accessed 2012 Aug 1. 77. Herrnkind W, Halusky J, Kanciruk P (1976) A Further Note on Phyllosoma
54. Tipper J (1979) Rarefaction and Rarefiction - The Use and Abuse of a Method Larvae Associated With Medusae. Bull MAR SCI 26: 110–112.
in Paleoecology. Paleobiology 5: 423–434. 78. Cox S, Johnston D (2003) Feeding biology of spiny lobster larvae and
55. Armougom F, Raoult D (2008) Use of pyrosequencing and DNA barcodes to implications for culture. Reviews in Fisheries Science 11: 89–106.
monitor variations in Firmicutes and Bacteroidetes communities in the gut 79. Johnston M, Johnston D, Knott B (2008) Ontogenetic Changes in the Structure
microbiota of obese humans. BMC genomics 9: 576–588. and Function of the Mouthparts and Foregut of Early and Late Stage Panulirus
56. Amaral-Zettler L, McCliment EA, Ducklow HW, Huse SM (2009) A method for ornatus (Fabricius, 1798) Phyllosomata (Decapoda: Palinuridae). J Crust Biol 28:
studying protistan diversity using massively parallel sequencing of V9 46–56.
hypervariable regions of small-subunit ribosomal RNA genes. PLoS ONE 4: 80. Tong L, Moss G, Paewai M, Pickering T (1997) Effect of brine-shrimp numbers
e6372. doi:10.1371/journal.pone.0006372. on growth and survival of early-stage phyllosoma larvae of the rock lobster Jasus
57. Bråte J, Logares R, Berney C, Ree D, Klaveness D, et al. (2010) Freshwater edwardsii. Mar Freshwater Res 48: 935–940.
Perkinsea and marine-freshwater colonizations revealed by pyrosequencing and 81. Sheppard SK, Harwood JD (2005) Advances in molecular ecology: tracking
phylogeny of environmental rDNA. ISME J 4: 1144–1153. trophic links through predator-prey food-webs. Funct Ecology 19: 751–762.
58. Fonseca VG, Carvalho GR, Sung W, Johnson HF, Power DM, et al. (2010) 82. Janssen J, Harbison GR (2009) Fish in Salps: the Association of Squaretails
Second-generation environmental sequencing unmasks marine metazoan (Tetragonurus Spp.) With Pelagic Tunicates. J Mar Biol Assoc UK 61: 917–927.
biodiversity. Nat Commun 1: 98. 83. Angel D (1991) Carbon flow within the colonial radiolarian microcosm.
59. Behnke A, Engel M, Christen R, Nebel M, Klein RR, et al. (2010) Depicting Symbiosis 10: 195–217.
more accurate pictures of protistan community complexity using pyrosequencing 84. Froneman PW, Pakhomov EA (1998) Trophic importance of the chaetognaths
of hypervariable SSU rRNA gene regions. Environ Microb 13: 340–349. Eukrohnia hamata and Sagitta gazellae in the pelagic system of the Prince
60. Metzker ML (2010) Sequencing technologies - the next generation. Nature Rev Edward Islands (Southern Ocean). Polar Biol 19: 242–249.
Gen 11: 31–46. 85. Baier CT, Purcell JE (1997) Effects of sampling and preservation on apparent
61. Valentini A, Miquel C, Nawaz MA, Bellemain E, Coissac E, et al. (2009) New feeding by chaetognaths. Mar Ecol Prog Ser 146: 37–42.
perspectives in diet analysis based on DNA barcoding and parallel pyrose- 86. Giesecke R, González H, Bathmann U (2010) The role of the chaetognath
quencing: the trnL approach. Mol Ecol Res 9: 51–60. Sagitta gazellae in the vertical carbon flux of the Southern Ocean. Polar Biol 33:
62. Soininen EM, Valentini A, Coissac E, Miquel C, Gielly L, et al. (2009) Analysing 293–304.
diet of small herbivores: the efficiency of DNA barcoding coupled with high- 87. Dilling L, Alldredge AL (1993) Can chaetognath fecal pellets contribute
throughput pyrosequencing for deciphering the composition of complex plant significantly to carbon flux? Mar Ecol Prog Ser 92: 51–58.
mixtures. Front Zool 6: 16. 88. Dilling L, Wilson J, Steinberg D, Alldredge A (1998) Feeding by the euphausiid
63. Prokopowich CD, Gregory TR, Crease TJ (2003) The correlation between Euphausia pacifica and the copepod Calanus pacificus on marine snow. Mar Ecol
rDNA copy number and genome size in eukaryotes. Genome 46: 48–50. Prog Ser 170: 189–201.
64. Suzuki MT, Giovannoni SJ (1996) Bias caused by template annealing in the 89. Turner JT (2002) Zooplankton fecal pellets, marine snow and sinking
amplification of mixtures of 16S rRNA genes by PCR. Appl Environ Microb 62: phytoplankton blooms. Aquat Microb Ecol 27: 57–102.
625–630. 90. Pomeroy LR, Hanson RB, McGillivary PA, Sherr BF, Kirchman D, et al. (1984)
65. Acinas SG, Sarma-Rupavtarm R, Klepac-Ceraj V, Polz MF (2005) PCR- Microbiology and Chemistry of Fecal Products of Pelagic Tunicates: Rates and
induced sequence artifacts and bias: insights from comparison of two 16S rRNA Fates. B Mar Sci 35: 426–439.
clone libraries constructed from the same sample. Appl Environ Microb 71: 91. Mochioka N, Iwamizu M (1996) Diet of anguilloid larvae: leptocephali feed
8966–8969. selectively on larvacean houses and fecal pellets. Mar Biol 125: 447–452.
66. Budowle B, Eisenberg AJ, van Daal A (2009) Low copy number typing has yet to 92. Terahara T, Chow S, Kurogi H, Lee S-H, Tsukamoto K, et al. (2011) Efficiency
achieve ‘‘general acceptance.’’ Forensic Sci Int: Gen Suppl Ser 2: 551–552. of peptide nucleic acid-directed PCR clamping and its application in the
67. van Oorschot R, Ballantyne K, Mitchell RJ (2010) Forensic trace DNA: a investigation of natural diets of the Japanese eel leptocephali. PLoS ONE 6:
review. Invest Genet 1: 1–17. e25715. doi:10.1371/journal.pone.0025715.

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