TY - JOUR
AU - Slater, John W.
AB - Abstract Establishing predator–prey relationships and determining competitive interactions within the plankton community remains a central aim of zooplankton ecology. Using bivalve larvae as a model system, a DNA-based dietary approach using general eukaryotic primers was evaluated. Prey DNA was preferentially amplified using a predator-specific endonuclease restriction enzyme and blocking primer. Application of the blocking primer in isolation resulted in 80% of recombinant clones carrying inserts of non-bivalve origin, increasing to 100% when combined with a restriction enzyme. Further validation was achieved using wild, naturally feeding larvae of Mysella spp. and Ostrea edulis. Of the sequenced clones, 75% originated from centric and pennate diatoms (Bacillariophyta). A further 16% originated from fungi representing the phyla Ascomycota and Basidiomycota. The remaining sequences belonged to flowering plants (Magnoliophyta), single-celled green algae (Prasinophyceae), potential parasites (Ichthyosporea), dinoflagellates (Dinophyceae) and brown algae (Phaeophyceae). No qualitative difference in diet was observed among these two particular species, although the diversity of prey observed suggests that this DNA-based approach is suitable for studying the trophic interactions of marine bivalve larvae. Furthermore, based on sequence alignments, slight modifications to the blocking primer sequence could adapt this basic approach to a wide diversity of consumers within the plankton community. INTRODUCTION Approximately 70% of all marine invertebrates have planktotrophic larvae, of which most are relatively small (<1 mm) (Thorson, 1950). The duration of exogenous feeding varies between species and is influenced by environmental factors such as salinity, temperature and food availability (Boidron-Métairon, 1995). Larvae feed on both heterotrophic and autotrophic organisms as well as detritus, while some species can directly utilize dissolved organic matter (Boidron-Métairon, 1995). The ability of larvae to adequately exploit their available food sources directly influences planktonic survival, metamorphosis and eventual recruitment to benthic populations (Olson and Olson, 1989). However, global climate change is altering phytoplankton phenology potentially disrupting the synchrony of meroplankton and their food sources (Hays et al., 2005). Despite the adult populations of many invertebrate species being of both ecological and commercial importance, little is known regarding specific trophic interactions of their larval stages, including feeding selectivity, ontogenetic shifts in prey preference or levels of feeding competition between various species. Such data are essential to understanding overall food web dynamics within the plankton community and predicting the effects a shifting plankton community structure may have on larval recruitment. Monitoring the predator–prey interactions within the plankton community remains difficult due to the small size of the organisms involved. Recently, dietary analyses using DNA-based techniques are providing insights into the trophic relationships for a diverse array of organisms (Deagle et al., 2009; Maloy et al., 2009; Riemann et al., 2010) and are potentially applicable to small larvae and zooplankton. Most applications are based on PCR amplification of gut contents using general primers targeting the 18S rRNA gene. Containing both conserved (for general primer design) and hypervariable (for phylogenetic comparison) regions, 18S rDNA allow for the simultaneous detection of a wide diversity of dietary organisms (Hillis and Dixon, 1991). However, this approach coamplifies the predator's DNA. In cases where gut contents can be readily isolated, amplification of predator DNA is generally a manageable concern (Barnard et al., 2006; Martin et al., 2006; Maloy et al., 2009; Riemann et al., 2010). This is in contrast to small larvae or zooplankton from which gut contents cannot be easily isolated. In such cases, DNA extracted from whole organisms results in a high concentration of predator DNA which will preferentially amplify over prey DNA when using a general primer approach (Polz and Cavanaugh, 1998). Therefore, a means to remove or inhibit the amplification of predator DNA is required in trophic studies within the plankton community when using general primers. Several strategies have been used to remove or inhibit predator/host DNA from PCR amplifications in dietary and disease studies. Predator-specific endonuclease restriction enzymes have been used to cut DNA before PCR amplification and/or prior to cloning. Working with scat samples from dolphins, Dunshea (Dunshea, 2009) was able to reduce the number of predator sequences in resulting clone libraries by up to 50% by restricting DNA prior to amplification and up to 87% with an additional restriction digest of the PCR products before cloning. Alternatively, blocking primers have been used to inhibit the amplification of predator sequences. Vestheim and Jarman (Vestheim and Jarman, 2008) demonstrated that their “annealing inhibiting blocker” was capable of completely eliminating predator amplifications (as detected by capillary electrophoresis) in mock samples with a 1000:1 ratio of predator-to-prey DNA. When applied to naturally feeding krill samples, 86% of sequences in subsequent clone libraries were of non-predator origin. Comparable results (76%) were obtained by Deagle et al. (Deagle et al., 2009) when using an annealing inhibiting blocking primer for analysis of fecal samples of seals. With a similar approach, Chow et al. (Chow et al., 2011) successfully used peptide nucleic acid-directed PCR clamping to suppress the amplification of predator DNA from gut content samples of larval Japanese spiny lobster. Of the sequenced clones, 93% carried inserts of non-predator origin. In this case, the phyllosoma larvae of the spiny lobster are relatively large (>15 mm) and the gut contents were crudely isolated prior to DNA extraction (Suzuki et al., 2008). A blocking approach has also been used to suppress the amplification of host DNA to aid in pathogen detection. In Troedsson et al. (Troedsson et al., 2008), the use of a peptide nucleic acid PCR hybridization blocking probe enhanced detection of parasite DNA by four orders of magnitude. To date, application of a general primer approach has not been applied to small (<1 mm) planktonic organisms. Using marine bivalve larvae as a model system, a combined annealing inhibiting blocker and specific endonuclease restriction approach was developed to provide a preliminary framework to explore qualitative similarities in diets between species. This approach was further validated using wild larvae of Mysella spp. and Ostrea edulis. METHOD Sample collection and larval sorting Larvae were collected from Lough Swilly, Ireland (Latitude: 55° 05′41.45″ N; Longitude: 7°31′43.19″ W), on 27 July 2010 using a vertical tow through the first 7 m of the water column with a 100 µm plankton net. Duration of the tow was held to a minimum to reduce the possibility of net feeding and all captured organisms were immediately preserved in 70% ethanol. Preserved samples were stored at 4°C until further processing. Subsamples of the preserved plankton were aliquoted into sterile Petri plates where bivalve larvae were isolated (using a pipette) and transferred to an additional Petri plate. Once a sufficient number of larvae were isolated, the entire group was rinsed in nuclease-free (NF) water and re-isolated to a clean Petri plate. This process continued until larvae were free of any visible (50×) non-bivalve material. At this point, larvae were further cleaned by soaking them in a 5% bleach solution for 5 min. Larvae were then rinsed (3×) in NF water and single larvae were transferred to sterile 1.5 mL microfuge tubes for DNA extraction. In addition, a 25 µL aliquot of the final rinse water was retained as a negative control in subsequent DNA extractions. As far as possible, larvae were collected, sorted, rinsed and prepared for DNA extraction using aseptic techniques. PCR: bivalve identification A large portion of the 18S rRNA gene (∼1250 bp) was amplified from each larval bivalve DNA (see below for extraction details) sample (n = 10) using the general eukaryotic primers E528F (Edgcomb et al., 2002) and EUKB (Medlin et al., 1988). Each 50 μL PCR reaction contained PCR buffer at 1×, 1.75 mM MgCl2, 0.8 μM of each primer, 200 μM of each deoxynucleoside triphosphate, 1.5 μM bovine serum albumin, 2.5 U Platinum Taq Polymerase (Invitrogen) and 3.0 μL of template DNA. Reactions were run under the following conditions: 5 min at 95°C followed by 35 cycles of 45 s denaturing (95°C), 45 s annealing (55°C), 1 min elongation (72°C) and an additional 10 min elongation at 72°C. PCR products were purified using a MinElute purification kit (Qiagen) and commercially sequenced (Beckman Coulter Genomics). Resulting sequence reads were assembled with Geneious Pro 5.0.4 (Drummond et al., 2009) and BLAST (Altschul et al., 1997) results were used to assign putative bivalve identifications to each larva. Blocking primer design and restriction site identification A near full-length 18S DNA sequence alignment was constructed for a diverse group of eukaryotic organisms including bivalves, potential bivalve prey and other organisms common in the plankton. Representative sequences (n = 110) were obtained from Genbank and aligned in Geneious Pro 5.1.4 (Drummond et al., 2009) using the MUSCLE algorithm. Within the alignment, two highly conserved regions were identified that allowed for the amplification of ∼225 bp region of variability. The forward primer 960F is a previously described eukaryotic-specific primer (Gast et al., 2004), while the reverse primer 1200sh (5′-catcacagacctgttattgc-3′) is the universal primer 1200R (Gast et al., 2004) shifted 6 bp. This shift was necessary to locate the primer closer to the variable region and allow more overlap with the bivalve-specific blocking primer BivBlk [5′-cctgttattgctccatctcgtgtggc-(3C)-3′]. The 5′ end of the blocking primer overlaps with the 3′ end of 1200sh, while its 3′ end anneals to an area conserved within bivalves but variable within their potential prey. In this sense, if BivBlk anneals to the bivalve template DNA first, it blocks the subsequent annealing of 1200sh. Polymerase elongation of BivBlk is arrested by modifying the 3′ terminus with a 3-carbon spacer molecule (3C) (Vestheim and Jarman, 2008). This transition between the hypervariable and conserved region of the 1200sh binding was the most suited for blocking primer design within the 18S region. In addition, within this 225 bp region, a DNA restriction enzyme was identified. Located within the bivalve-specific region of the blocking primer, the enzyme BssSI will cleave bivalve DNA (or PCR products) while leaving potential prey unrestricted (Fig. 1). Fig. 1. Open in new tabDownload slide Partial alignment of 18S rRNA gene sequences from a representative sample of bivalves, potential protist prey and other planktonic consumers. Ostrea edulis served as a reference sequence to which all others were aligned. Shaded regions depict the location and arrangement of the amplification primers (960F and 1200sh), blocking primer (BivBlk) and BssSI restriction site. See online supplementary data for a color version of this figure. Fig. 1. Open in new tabDownload slide Partial alignment of 18S rRNA gene sequences from a representative sample of bivalves, potential protist prey and other planktonic consumers. Ostrea edulis served as a reference sequence to which all others were aligned. Shaded regions depict the location and arrangement of the amplification primers (960F and 1200sh), blocking primer (BivBlk) and BssSI restriction site. See online supplementary data for a color version of this figure. Blocking primer and restriction digest evaluation To determine the effectiveness of a blocking primer and/or restriction enzyme to repress the amplification of bivalve DNA while allowing DNA signatures originating from gut contents to be detected, an experimental design was established to evaluate each approach. To start, DNA was extracted from individual bivalve larvae and each was identified based on their 18S rRNA gene sequence (see below). To obtain sufficient DNA for the experimental protocol, DNA from three separate larvae of the same species (Mysella spp.) were pooled. This pooled DNA was then split into two separate fractions, one of which was digested with BssSI. Each fraction was PCR amplified with and without the use of a blocking primer, thus creating four sets of PCR products. Half of the PCR products from each set were directly cloned. The remaining PCR products were subjected to a final round of BssSI restriction digest prior to cloning. In all, eight separate clone libraries were created (Fig. 2). PCR and BssSI restriction conditions were as described below. Fig. 2. Open in new tabDownload slide Diagram depicting the work flow used to develop a molecular approach to the dietary analysis of bivalve larvae. In CL-1 and CL-5, DNA was directly amplified with and without the use of a blocking primer to assess its potential to suppress the amplification of the bivalve DNA. In CL-2 and CL-6, a subsequent round of BssSI restriction digest was used to remove bivalve sequences post-amplification. In CL-3 and CL-7, a BssSI restriction digest was performed to assess its ability to remove bivalve template DNA prior to PCR amplification with and without the use of a blocking primer. In CL-4 and CL-8, a second BssSI restriction digest was performed on the resulting PCR products prior to cloning. Ratios indicate the number of clones carrying bivalve sequences relative to those of non-bivalve origin. Fig. 2. Open in new tabDownload slide Diagram depicting the work flow used to develop a molecular approach to the dietary analysis of bivalve larvae. In CL-1 and CL-5, DNA was directly amplified with and without the use of a blocking primer to assess its potential to suppress the amplification of the bivalve DNA. In CL-2 and CL-6, a subsequent round of BssSI restriction digest was used to remove bivalve sequences post-amplification. In CL-3 and CL-7, a BssSI restriction digest was performed to assess its ability to remove bivalve template DNA prior to PCR amplification with and without the use of a blocking primer. In CL-4 and CL-8, a second BssSI restriction digest was performed on the resulting PCR products prior to cloning. Ratios indicate the number of clones carrying bivalve sequences relative to those of non-bivalve origin. DNA extraction and BssSI restriction digest DNA was extracted from single larvae (n = 7) using the QIAamp DNA Micro Kit (Qiagen) following the manufacturer's' tissue protocol. In addition to rinse water-negative controls, a blank sample consisting only of DNA extraction reagents was also included. The initial proteinase K digestion was run overnight and carrier RNA was added to aid in DNA recovery. From the final 25 µL elution, a 5 µL aliquot was removed and used to identify each bivalve as described above. BssSI restriction digest was used on both genomic DNA and PCR products. In each case, digests contained: DNA or PCR products, 1× buffer #3, 1× bovine serum albumin and 5 U BssSI restriction enzyme (New England Biolabs). All DNA and restriction digest steps were undertaken in a UV-sterilized laminar flow cabinet to prevent contamination. PCR: gut content identification A portion of the 18S rRNA gene (∼225 bp) was amplified from each DNA sample using the eukaryotic-specific primer 960F (Gast et al., 2004; Pechenik et al., 2004) and universal primer 1200sh (5′-catcacagacctgttattgc-3′). Where indicated in the cloning experiment and in dietary assessment of wild Mysella spp. and O. edulis, the blocking primer BivBlk [5′-cctgttattgctccatctcgtgtggc-(3C)-3′] was also included. Each 50 μL PCR reaction contained PCR buffer at 1×, 1.5 mM MgCl2, 0.12 μM of 960F and 1200sh, 1.2 μM BivBlk, 200 μM of each deoxynucleoside triphosphate, 1.5 μM bovine serum albumin, 2.5 U HotStarTaq DNA Polymerase (Qiagen) and 3 μL of template DNA. PCR reaction conditions were as stated above. All PCR reactions were set up in a separate UV-sterilized laminar flow cabinet solely dedicated to PCR preparation. PCR products or subsequent restriction products were purified using a MinElute Purification Kit (Qiagen) from which 3 μL was cloned using a TOPO-TA (Invitrogen) cloning kit. Recombinant clones were screened to ensure that they were carrying inserts of the correct size prior to purification using a QIAprep Miniprep kit (Qiagen). Approximately 20 purified plasmids from each bivalve larva were commercially sequenced using the primer 960F and 1200sh. Sequence analysis, BLAST identification and statistical analysis All sequences were assembled and trimmed of remaining vector sequence using Geneious Pro 5.1.4. Each unique sequence type, those differing by ≥1 bp, was BLASTed against the nr/nt data set hosted by the National Center of Biotechnology Information (NCBI). BLAST-based identifications were used to identify nearest neighbor sequences for inclusion in phylogenetic analysis to determine phylum level taxonomic affiliations using an unrooted neighbor-joining tree with Tamura–Nei distances. A consensus of the top matched organisms was used to determine the lowest level of taxonomic resolution based on the NCBI's Taxonomy Browser nomenclature. RESULTS Bivalve identification Amplifying a large portion of the 18S rRNA gene provided a convenient and reliable way to identify individual larvae independent of morphology. BLAST-based sequence similarities were high (99–100%), suggesting that larvae were either Mysella vitrea or O. edulis (Table I). However, M. vitrea is native to Australia and has not previously been reported in Irish waters. Alternatively, the sequences obtained here could belong to Mysella bidentata, a related species common to the region but lacking sequence entries in public databases. As this cannot be reconciled in the current study, all references are reported as Mysella spp. In this case, both were morphologically dissimilar. Mysella spp. (shell height 275.0 ± 58.8 µm) were a pale beige/white in color, flattened ovals in shape with valves that appeared relatively smooth. In contrast, O. edulis (shell height 187.0 ± 23.1 µm) were darker in color, more spherical in shape and had valves with a rougher looking texture. Negative controls for larval rinse water, DNA extraction reagents and PCR reagents produce no visible PCR products after amplification for bivalve identification or gut content analysis. Table I: Origin, size and larval identity of bivalves Sample ID . Shell height (µm) . Closest match BLAST . Percent similarity . Accession number . Larvae used in develop of the molecular approach  LB-493 259.5 Mysella vitrea 99 HQ719240  LB-498 165.7 Mysella vitrea 99 HQ719239  LB-504 285.6 Mysella vitrea 99 HQ719238 Larvae used in comparative feeding experiment  LB-481 340.7 Mysella vitrea 99 HQ719247  LB-483 252.7 Mysella vitrea 99 HQ719246  LB-485 231.7 Mysella vitrea 99 HQ719245  LB-486 162.6 Ostrea edulis 99 HQ719244  LB-487 185.9 Ostrea edulis 100 HQ719243  LB-488 184.6 Ostrea edulis 100 HQ719242  LB-489 218.8 Ostrea edulis 100 HQ719241 Sample ID . Shell height (µm) . Closest match BLAST . Percent similarity . Accession number . Larvae used in develop of the molecular approach  LB-493 259.5 Mysella vitrea 99 HQ719240  LB-498 165.7 Mysella vitrea 99 HQ719239  LB-504 285.6 Mysella vitrea 99 HQ719238 Larvae used in comparative feeding experiment  LB-481 340.7 Mysella vitrea 99 HQ719247  LB-483 252.7 Mysella vitrea 99 HQ719246  LB-485 231.7 Mysella vitrea 99 HQ719245  LB-486 162.6 Ostrea edulis 99 HQ719244  LB-487 185.9 Ostrea edulis 100 HQ719243  LB-488 184.6 Ostrea edulis 100 HQ719242  LB-489 218.8 Ostrea edulis 100 HQ719241 Open in new tab Table I: Origin, size and larval identity of bivalves Sample ID . Shell height (µm) . Closest match BLAST . Percent similarity . Accession number . Larvae used in develop of the molecular approach  LB-493 259.5 Mysella vitrea 99 HQ719240  LB-498 165.7 Mysella vitrea 99 HQ719239  LB-504 285.6 Mysella vitrea 99 HQ719238 Larvae used in comparative feeding experiment  LB-481 340.7 Mysella vitrea 99 HQ719247  LB-483 252.7 Mysella vitrea 99 HQ719246  LB-485 231.7 Mysella vitrea 99 HQ719245  LB-486 162.6 Ostrea edulis 99 HQ719244  LB-487 185.9 Ostrea edulis 100 HQ719243  LB-488 184.6 Ostrea edulis 100 HQ719242  LB-489 218.8 Ostrea edulis 100 HQ719241 Sample ID . Shell height (µm) . Closest match BLAST . Percent similarity . Accession number . Larvae used in develop of the molecular approach  LB-493 259.5 Mysella vitrea 99 HQ719240  LB-498 165.7 Mysella vitrea 99 HQ719239  LB-504 285.6 Mysella vitrea 99 HQ719238 Larvae used in comparative feeding experiment  LB-481 340.7 Mysella vitrea 99 HQ719247  LB-483 252.7 Mysella vitrea 99 HQ719246  LB-485 231.7 Mysella vitrea 99 HQ719245  LB-486 162.6 Ostrea edulis 99 HQ719244  LB-487 185.9 Ostrea edulis 100 HQ719243  LB-488 184.6 Ostrea edulis 100 HQ719242  LB-489 218.8 Ostrea edulis 100 HQ719241 Open in new tab Blocking primer and BssSI restriction To assess the effectiveness of the blocking primer, BssSI restriction digest or a combined approach to suppress the amplification of bivalve DNA, a total of 10 clones from each of the eight clone libraries (CL) were sequenced (Table II, Fig. 2). PCR products used in CL 1–4 were obtained without the use of a blocking primer, while the blocking primer was used to obtain PCR products used in CL 5–7. PCR products in CL-1 were obtained without the use of BssSI restriction enzyme and contained nine bivalve sequences with the remaining one sequence of non-bivalve origin. A further BssSI restriction of these PCR products resulted in CL-2 containing eight bivalve and two non-bivalve sequences. BssSI restriction digest of DNA prior to amplification (CL-3) resulted in 10 clones carrying inserts of bivalve origin. In contrast, an additional BssSI restriction of these PCR products resulted in CL-4 containing two sequences of bivalve and eight of non-bivalve origin. CL-5 was constructed after amplification with the blocking primer. In this instance, two sequences were of bivalve origin, while the remaining eight were non-bivalve. Combining the use of the blocking primer with BssSI restriction digest(s) increased the number of non-bivalve sequences recovered to 10. This was irrespective as to whether DNA was digested after amplification (CL-6), the PCR products were digested prior to amplification (CL-7), or a BssSI digest was used both before and after amplification (CL-8). Table II: Results of the cloning experiment evaluating the effectiveness of a selective blocking primer and restriction enzyme to suppress the amplification of bivalve DNA Clone library . Sequence origin . (Number of sequences recovered) Closest BLAST match; percent similarity . Accession number . Bivalve . Non-bivalve . 1 9 1 (9) Mysella vitrea, specimen voucher BMNH 20070233; 100% HQ7193456–HQ719465 (1) Stephanodiscus hantzschii isolate UTCC 267; 100% 2 8 2 (8) Mysella vitrea, specimen voucher BMNH 20070233; 99–100% HQ719446–HQ719455 (2) Stephanodiscus hantzschii isolate UTCC 267; 100% 3 10 0 (10) Mysella vitrea, specimen voucher BMNH 20070233; 99–100% HQ719436–HQ719445 4 2 8 (2) Mysella vitrea, specimen voucher BMNH 20070233; 100% HQ719426–HQ719435 (8) Stephanodiscus hantzschii isolate UTCC 267; 96–100% 5 2 8 (2) Mysella vitrea, specimen voucher BMNH 20070233; 100% HQ719416–HQ719425 (8) Stephanodiscus hantzschii isolate UTCC 267; 100% 6 0 10 (10) Stephanodiscus hantzschii isolate UTCC 267; 99–100% HQ719406–HQ719415 7 0 10 (10) Stephanodiscus hantzschii isolate UTCC 267; 99–100% HQ719396–HQ719405 8 0 10 (10) Stephanodiscus hantzschii isolate UTCC 267; 99–100% HQ719386–HQ719395 Clone library . Sequence origin . (Number of sequences recovered) Closest BLAST match; percent similarity . Accession number . Bivalve . Non-bivalve . 1 9 1 (9) Mysella vitrea, specimen voucher BMNH 20070233; 100% HQ7193456–HQ719465 (1) Stephanodiscus hantzschii isolate UTCC 267; 100% 2 8 2 (8) Mysella vitrea, specimen voucher BMNH 20070233; 99–100% HQ719446–HQ719455 (2) Stephanodiscus hantzschii isolate UTCC 267; 100% 3 10 0 (10) Mysella vitrea, specimen voucher BMNH 20070233; 99–100% HQ719436–HQ719445 4 2 8 (2) Mysella vitrea, specimen voucher BMNH 20070233; 100% HQ719426–HQ719435 (8) Stephanodiscus hantzschii isolate UTCC 267; 96–100% 5 2 8 (2) Mysella vitrea, specimen voucher BMNH 20070233; 100% HQ719416–HQ719425 (8) Stephanodiscus hantzschii isolate UTCC 267; 100% 6 0 10 (10) Stephanodiscus hantzschii isolate UTCC 267; 99–100% HQ719406–HQ719415 7 0 10 (10) Stephanodiscus hantzschii isolate UTCC 267; 99–100% HQ719396–HQ719405 8 0 10 (10) Stephanodiscus hantzschii isolate UTCC 267; 99–100% HQ719386–HQ719395 Clone library numbers correspond to those depicted in Fig. 2. Open in new tab Table II: Results of the cloning experiment evaluating the effectiveness of a selective blocking primer and restriction enzyme to suppress the amplification of bivalve DNA Clone library . Sequence origin . (Number of sequences recovered) Closest BLAST match; percent similarity . Accession number . Bivalve . Non-bivalve . 1 9 1 (9) Mysella vitrea, specimen voucher BMNH 20070233; 100% HQ7193456–HQ719465 (1) Stephanodiscus hantzschii isolate UTCC 267; 100% 2 8 2 (8) Mysella vitrea, specimen voucher BMNH 20070233; 99–100% HQ719446–HQ719455 (2) Stephanodiscus hantzschii isolate UTCC 267; 100% 3 10 0 (10) Mysella vitrea, specimen voucher BMNH 20070233; 99–100% HQ719436–HQ719445 4 2 8 (2) Mysella vitrea, specimen voucher BMNH 20070233; 100% HQ719426–HQ719435 (8) Stephanodiscus hantzschii isolate UTCC 267; 96–100% 5 2 8 (2) Mysella vitrea, specimen voucher BMNH 20070233; 100% HQ719416–HQ719425 (8) Stephanodiscus hantzschii isolate UTCC 267; 100% 6 0 10 (10) Stephanodiscus hantzschii isolate UTCC 267; 99–100% HQ719406–HQ719415 7 0 10 (10) Stephanodiscus hantzschii isolate UTCC 267; 99–100% HQ719396–HQ719405 8 0 10 (10) Stephanodiscus hantzschii isolate UTCC 267; 99–100% HQ719386–HQ719395 Clone library . Sequence origin . (Number of sequences recovered) Closest BLAST match; percent similarity . Accession number . Bivalve . Non-bivalve . 1 9 1 (9) Mysella vitrea, specimen voucher BMNH 20070233; 100% HQ7193456–HQ719465 (1) Stephanodiscus hantzschii isolate UTCC 267; 100% 2 8 2 (8) Mysella vitrea, specimen voucher BMNH 20070233; 99–100% HQ719446–HQ719455 (2) Stephanodiscus hantzschii isolate UTCC 267; 100% 3 10 0 (10) Mysella vitrea, specimen voucher BMNH 20070233; 99–100% HQ719436–HQ719445 4 2 8 (2) Mysella vitrea, specimen voucher BMNH 20070233; 100% HQ719426–HQ719435 (8) Stephanodiscus hantzschii isolate UTCC 267; 96–100% 5 2 8 (2) Mysella vitrea, specimen voucher BMNH 20070233; 100% HQ719416–HQ719425 (8) Stephanodiscus hantzschii isolate UTCC 267; 100% 6 0 10 (10) Stephanodiscus hantzschii isolate UTCC 267; 99–100% HQ719406–HQ719415 7 0 10 (10) Stephanodiscus hantzschii isolate UTCC 267; 99–100% HQ719396–HQ719405 8 0 10 (10) Stephanodiscus hantzschii isolate UTCC 267; 99–100% HQ719386–HQ719395 Clone library numbers correspond to those depicted in Fig. 2. Open in new tab Larval diet analysis To validate the use of a restriction enzyme and blocking primer approach with wild samples, ∼20 sequences were obtained from each of seven larvae (3, Mysella spp.; 4, O. edulis). Within the 138 sequences recovered (Genbank accession numbers HQ719248–HQ719385), 26 unique sequence types were identified representing eight separate phyla (Fig. 3). Bacillariophyta were the most commonly observed organisms representing 76.1% of total sequences recovered, and also contained the highest diversity with 13 unique sequences. This was followed by the Ascomycota which accounted for 13.8% of the total and was composed of four unique sequences. The Streptophyta (3.6%) and Basidiomycota (2.2%) accounted for an additional two and three unique sequences, respectively. The remaining phyla, Heterokontophycophyta, Chlorophyta, Dinophyceae and Ichthyosporea, each accounted for ≤1.4% of the total and were represented by a single unique sequence type (Fig. 4). The overall taxonomic resolution varied, but sequences could generally be assigned to family groups with confidence (Table III). Table III: Classification results of the 26 unique sequence types recovered from Ostrea edulis and Mysella spp. larvae Unique sequence ID . Total sequences recovered . Higher taxon (Phylum) . Similarity (%) . Lowest consensus taxon . Lowest classification level . US-1 1 Bacillariophyta 99 Thalassiosiraceae Family US-2 56 Bacillariophyta 100 Thalassiosiraceae Family US-3 1 Bacillariophyta 99 Thalassiosiraceae Family US-4 2 Bacillariophyta 99 Thalassiosira Genus US-5 1 Bacillariophyta 99 Thalassiosiraceae Family US-6 4 Bacillariophyta 100 Thalassiosiraceae Family US-7 25 Bacillariophyta 94–95 Coscinodiscophyceae Class US-8 1 Bacillariophyta 97–98 Pleurosigmataceae Family US-9 1 Bacillariophyta 97–98 Pleurosigmataceae Family US-10 7 Bacillariophyta 97–98 Pleurosigmataceae Family US-11 2 Bacillariophyta 99 Navicula Genus US-12 3 Bacillariophyta 98–100 Cymatosiraceae Family US-13 1 Bacillariophyta 99–100 Cymatosiraceae Family US-14 1 Heterokontophycophyta 100 Ectocarpales Order US-15 1 Streptophyta 100 Magnoliophyta Class US-16 4 Streptophyta 100 Magnoliophyta Class US-17 2 Chlorophyta 100 Pycnococcaceae Family US-18 1 Dinophyceae ≤94 Unknown NA US-19 2 Ichthyosporea ≤94 Unknown NA US-20 1 Basidiomycota 99 Agaricomycetes Class US-21 1 Basidiomycota 99 Agaricomycetes Class US-22 1 Basidiomycota 100 Tremellaceae Family US-23 5 Ascomycota 100 Mycosphaerellaceae Family US-24 4 Ascomycota 100 Hypocreales Order US-25 1 Ascomycota 100 Trichocomaceae Family US-26 9 Ascomycota 99 Dipodascaceae Family Unique sequence ID . Total sequences recovered . Higher taxon (Phylum) . Similarity (%) . Lowest consensus taxon . Lowest classification level . US-1 1 Bacillariophyta 99 Thalassiosiraceae Family US-2 56 Bacillariophyta 100 Thalassiosiraceae Family US-3 1 Bacillariophyta 99 Thalassiosiraceae Family US-4 2 Bacillariophyta 99 Thalassiosira Genus US-5 1 Bacillariophyta 99 Thalassiosiraceae Family US-6 4 Bacillariophyta 100 Thalassiosiraceae Family US-7 25 Bacillariophyta 94–95 Coscinodiscophyceae Class US-8 1 Bacillariophyta 97–98 Pleurosigmataceae Family US-9 1 Bacillariophyta 97–98 Pleurosigmataceae Family US-10 7 Bacillariophyta 97–98 Pleurosigmataceae Family US-11 2 Bacillariophyta 99 Navicula Genus US-12 3 Bacillariophyta 98–100 Cymatosiraceae Family US-13 1 Bacillariophyta 99–100 Cymatosiraceae Family US-14 1 Heterokontophycophyta 100 Ectocarpales Order US-15 1 Streptophyta 100 Magnoliophyta Class US-16 4 Streptophyta 100 Magnoliophyta Class US-17 2 Chlorophyta 100 Pycnococcaceae Family US-18 1 Dinophyceae ≤94 Unknown NA US-19 2 Ichthyosporea ≤94 Unknown NA US-20 1 Basidiomycota 99 Agaricomycetes Class US-21 1 Basidiomycota 99 Agaricomycetes Class US-22 1 Basidiomycota 100 Tremellaceae Family US-23 5 Ascomycota 100 Mycosphaerellaceae Family US-24 4 Ascomycota 100 Hypocreales Order US-25 1 Ascomycota 100 Trichocomaceae Family US-26 9 Ascomycota 99 Dipodascaceae Family Placement into high-level taxons was based on phylogenetic analysis, while low-level consensus taxons were determined using BLAST results. Open in new tab Table III: Classification results of the 26 unique sequence types recovered from Ostrea edulis and Mysella spp. larvae Unique sequence ID . Total sequences recovered . Higher taxon (Phylum) . Similarity (%) . Lowest consensus taxon . Lowest classification level . US-1 1 Bacillariophyta 99 Thalassiosiraceae Family US-2 56 Bacillariophyta 100 Thalassiosiraceae Family US-3 1 Bacillariophyta 99 Thalassiosiraceae Family US-4 2 Bacillariophyta 99 Thalassiosira Genus US-5 1 Bacillariophyta 99 Thalassiosiraceae Family US-6 4 Bacillariophyta 100 Thalassiosiraceae Family US-7 25 Bacillariophyta 94–95 Coscinodiscophyceae Class US-8 1 Bacillariophyta 97–98 Pleurosigmataceae Family US-9 1 Bacillariophyta 97–98 Pleurosigmataceae Family US-10 7 Bacillariophyta 97–98 Pleurosigmataceae Family US-11 2 Bacillariophyta 99 Navicula Genus US-12 3 Bacillariophyta 98–100 Cymatosiraceae Family US-13 1 Bacillariophyta 99–100 Cymatosiraceae Family US-14 1 Heterokontophycophyta 100 Ectocarpales Order US-15 1 Streptophyta 100 Magnoliophyta Class US-16 4 Streptophyta 100 Magnoliophyta Class US-17 2 Chlorophyta 100 Pycnococcaceae Family US-18 1 Dinophyceae ≤94 Unknown NA US-19 2 Ichthyosporea ≤94 Unknown NA US-20 1 Basidiomycota 99 Agaricomycetes Class US-21 1 Basidiomycota 99 Agaricomycetes Class US-22 1 Basidiomycota 100 Tremellaceae Family US-23 5 Ascomycota 100 Mycosphaerellaceae Family US-24 4 Ascomycota 100 Hypocreales Order US-25 1 Ascomycota 100 Trichocomaceae Family US-26 9 Ascomycota 99 Dipodascaceae Family Unique sequence ID . Total sequences recovered . Higher taxon (Phylum) . Similarity (%) . Lowest consensus taxon . Lowest classification level . US-1 1 Bacillariophyta 99 Thalassiosiraceae Family US-2 56 Bacillariophyta 100 Thalassiosiraceae Family US-3 1 Bacillariophyta 99 Thalassiosiraceae Family US-4 2 Bacillariophyta 99 Thalassiosira Genus US-5 1 Bacillariophyta 99 Thalassiosiraceae Family US-6 4 Bacillariophyta 100 Thalassiosiraceae Family US-7 25 Bacillariophyta 94–95 Coscinodiscophyceae Class US-8 1 Bacillariophyta 97–98 Pleurosigmataceae Family US-9 1 Bacillariophyta 97–98 Pleurosigmataceae Family US-10 7 Bacillariophyta 97–98 Pleurosigmataceae Family US-11 2 Bacillariophyta 99 Navicula Genus US-12 3 Bacillariophyta 98–100 Cymatosiraceae Family US-13 1 Bacillariophyta 99–100 Cymatosiraceae Family US-14 1 Heterokontophycophyta 100 Ectocarpales Order US-15 1 Streptophyta 100 Magnoliophyta Class US-16 4 Streptophyta 100 Magnoliophyta Class US-17 2 Chlorophyta 100 Pycnococcaceae Family US-18 1 Dinophyceae ≤94 Unknown NA US-19 2 Ichthyosporea ≤94 Unknown NA US-20 1 Basidiomycota 99 Agaricomycetes Class US-21 1 Basidiomycota 99 Agaricomycetes Class US-22 1 Basidiomycota 100 Tremellaceae Family US-23 5 Ascomycota 100 Mycosphaerellaceae Family US-24 4 Ascomycota 100 Hypocreales Order US-25 1 Ascomycota 100 Trichocomaceae Family US-26 9 Ascomycota 99 Dipodascaceae Family Placement into high-level taxons was based on phylogenetic analysis, while low-level consensus taxons were determined using BLAST results. Open in new tab Fig. 3. Open in new tabDownload slide High-level phylogenetic relationship of 138 dietary sequences obtained from O. edulis and Mysella spp. based on an unrooted neighbor-joining tree calculated with Tamura–Nei distances. Each unique sequence type (US-#) is followed by the total number of sequences obtained from O. edulis and Mysella spp. Related organisms are followed by GenBank accession numbers. Fig. 3. Open in new tabDownload slide High-level phylogenetic relationship of 138 dietary sequences obtained from O. edulis and Mysella spp. based on an unrooted neighbor-joining tree calculated with Tamura–Nei distances. Each unique sequence type (US-#) is followed by the total number of sequences obtained from O. edulis and Mysella spp. Related organisms are followed by GenBank accession numbers. Fig. 4. Open in new tabDownload slide Occurrence frequencies of the eight phyla represented in the dietary sequences recovered from O. edulis and Mysella spp. larvae. Fig. 4. Open in new tabDownload slide Occurrence frequencies of the eight phyla represented in the dietary sequences recovered from O. edulis and Mysella spp. larvae. Further qualitative dietary comparisons were made at the level of bivalve species. ANOSIM results showed no difference in the dietary similarity between the two species (R = 0.056, P = 0.343). Likewise, no difference was observed after removal of the fungal sequences (R = 0.120, P = 0.257) or analysis of only the diatom sequences (R = 0.111, P = 0.257). Comparing the general dietary patterns, one main trend is apparent. A greater number (18) and unique (6) fungal sequence types were recovered from O. edulis when compared with Mysella spp. Only four fungal sequences were recovered from Mysella spp. larvae, each representing the same sequence. DISCUSSION The successful detection of dietary elements from larval bivalves demonstrates the utility of an annealing inhibiting blocking primer and predator-specific restriction enzyme approach for trophic investigations within planktonic food webs. In previous studies using predator-specific restriction digests (Dunshea, 2009) and blocking primers (Vestheim and Jarman, 2008; Deagle et al., 2009; Chow et al., 2011), amplification of predator DNA was reduced to acceptable levels, although not completely eliminated. In those studies, DNA was obtained from either fecal samples or isolated gut contents. In contrast, the small size (<350 µm) of bivalve larvae used here required that DNA be extracted from the whole organism. The resulting ratio of predator-to-prey DNA, although not empirically measured, was presumably very high. Eight separate clone libraries were constructed to assess the relative ability of a BssSI restriction digest and/or blocking primer to eliminate the amplification of bivalve DNA (Fig. 2). Not surprisingly, CL 1–3 constructed without the use of a blocking primer largely contained predator sequences. In contrast to the results obtained by Dunshea (Dunshea, 2009), the use of a single BssSI digest, either before or after amplifications had little effect on the results. However, there is one exception. CL-4 contained only two predator sequences with the remaining eight from a non-bivalve origin. In this instance, a BssSI restriction digest was used before and after amplification. The drawback here was the low ligation efficiency, requiring hundreds of clones to be screened to find a suitable number carrying PCR product inserts. More promising results were obtained in CL 5–8 constructed with the BivBlk blocking primer. The use of the blocking primer alone gave comparable results to those previously reported (Vestheim and Jarman, 2008; Deagle et al., 2009), with eight of sequences from a non-bivalve origin. When combined with a BssSI restriction digest, all 10 of the cloned sequences could be attributed to a non-bivalve origin. The use of a BssSI restriction digest before amplification and/or after amplification had no observable effects on the results. Therefore, the approach used in CL-7, a BssSI restriction digest prior to amplification with the blocking primer BivBlk, was adopted for further validation with wild samples. When applied to naturally feeding larvae of wild Mysella spp. and O. edulis, only 1.4% of the 138 sequences originated from the bivalves themselves. This represents an improvement over previously reported values when using a specific restriction enzyme or blocking primer in isolation (Vestheim and Jarman, 2008; Deagle et al., 2009; Dunshea, 2009). The remaining non-bivalve sequences were predominantly diatoms. Diatom sequences accounted for 76.1% of the total, in which 13 unique sequence types were observed (Fig. 3) belonging to both centric (Coscinodiscophyceae) and pennate diatoms (Bacillariophyceaea). Six of these unique sequence types belonged to the family Thalassiosiraceae, but only one (Thallassiosira) could be resolved to the genus level. An additional centric diatom sequence type belonged to the family Cymatosiraceaea, while the remaining one could not be resolved beyond centric diatom due to low sequence homology. Taxonomic resolution of pennate diatoms was slightly better with the one belonging to the genus Pleurosigma and the other to Navicula. Previous studies of marine bivalves have also shown the dietary importance of diatoms (Fritz et al., 1984) and they are routinely used in commercial diet formulations (Gosling, 2003). However, field studies and those using natural plankton assemblages have not reported on the taxonomic identity of ingested prey to any great degree. Earlier work has largely been concerned with prey size distributions and generically grouped prey as heterotrophs, autotrophs, centric or pennate diatoms, dinoflagellates etc. This lack of taxonomic resolution is a reflection of the methodologies being used. Removal experiments, based on microscopically characterizing a plankton assemblage before and after the addition of larva (Fritz et al., 1984), are time-consuming and require a skilled taxonomist. Removal experiments using a flow cytometer (Baldwin, 1995; Baldwin and Newell, 1995) identify prey based on size distributions, as do radio isotope uptake studies of size-fractionated plankton samples (Baldwin and Newell, 1991). Epifluorescence microscopy of larval gut contents (Raby et al., 1997) provides size distributions and a crude level of taxonomic information, but is limited to autotrophic organisms only. Although with the approach presented here, it was not possible to resolve prey taxonomy to the level of species, the level achieved for the diatoms represents an improvement over previously published methods. Taxonomic resolution for the 33 non-diatom sequences was somewhat less than that of the diatoms (Fig. 3). The majority of these belonged to Fungi and contained seven unique sequences, four of which could be resolved to the Family level, while the remaining three could be placed at only the Class level. Similarly, within the three unique sequences attributed to the Plantae, the single-celled Chlorophyta could be resolved to the family (Pycnococcaceae) level, while those belonging to flowering plants (presumably originating from pollen grains) could only be placed within the Class Magnoliophyta. Additionally, one sequence was recovered that belonged to a brown macro alga of the Order Ectocarples. The final non-diatom sequences were of low homology and could only be attributed to organisms belonging to the Dinophyceae and Ichthyosporea. While fungal sequences have been reported in the diets of lobster larvae (Suzuki et al., 2008; Chow et al., 2011), it is not readily apparent if they represent a portion of the bivalve diet. Each bivalve larva used here was visually inspected (50×) and soaked in a 5% bleach solution to reduce the chances of introducing non-dietary organisms into DNA extractions. Despite these precautions, homogenization of whole larvae may have introduced DNA from organisms associated with the outer valve surface. Two observations support this assertion. Individual larvae tended to be associated with a single fungal sequence type, possibly suggesting their valves were colonized by a specific fungus. Furthermore, the majority (82%) of fungal sequences were associated with O. edulis larvae. The larval shell of O. edulis is more textured than that of the smooth-shelled Mysella spp. As such, the rinsing procedures and bleaching step may have been less effective at removing valve-associated organisms from O. edulis. Pollen grains, on the other hand, have not been previously reported in the diets of larval bivalves. Even within dietary analyses of adult bivalves, only a rare passing reference is made to pollen grains as a potential food source (Shumway et al., 1987). Only one comparative study has been reported from wild naturally feeding larvae. Using epifluorescence microscopy, Raby et al. (Raby et al., 1997) demonstrated a size-dependent partitioning of food resources between similarly sized M. edulis and M. arenaria. Based on the 26 unique sequences observed here, there were no qualitative differences in the diets of O. edulis and Mysella spp. This does, however, represent an important step forward in analyzing the diets of bivalve larvae and provides an initial view of the general taxonomic composition of gut contents. A detailed knowledge of the taxonomic associations of dietary items is necessary for directing the development of more robust general primers or identifying specific organisms for use in quantitative PCR studies. Further refinements of the technique will improve taxonomic resolution and dietary diversity measures. There are several challenges associated with using universal primers to track predator–prey interactions within the plankton. As was seen in this study, taxonomic resolution can vary between groups of organisms, and is dependent on both the region of DNA being amplified and its overall length of variability. Although the 18S rRNA gene is widely used in phylogenetic studies, it is not universally suitable. For example, 18S rDNA sequences are too conserved for the discrimination of most fungal groups and molecular identification to the species level is dependent on sequencing of the ITS1-rDNA region (Begerow et al., 2010). On the other hand, 18S rDNA sequences are widely used for phylogenetic analysis and species level identification of protists (Duff et al., 2008). Most phylogenetic studies and/or DNA barcoding applications use a minimum of 650 bp sequence (Hajibabaei et al., 2007; Duff et al., 2008). This is in contrast to dietary studies in which the length of amplification products is limited due to the rapid degradation of gut content DNA (Deagle et al., 2006; Simonelli et al., 2009; Troedsson et al., 2009). In this respect, a tradeoff exists between sensitivity (greater chance of detecting shorter fragments) and taxonomic resolution (more data contained in longer sequences). The 225 bp fragment of 18S rDNA amplified in this study did not provide species level identification within the diatoms. It is likely that both the relatively short sequences used and a poor representation of related organisms within representative databases limited the level of taxonomic resolution. Given the wide diversity of potential prey organisms within the plankton community, it is unlikely that a single genetic marker would be sufficient to capture all possible dietary diversity. In the absence of a truly universal marker, 18S rDNA remains a logical choice for dietary analysis among planktonic consumers in which the majority of their perceived diet is protists. Based on sequence alignments (Fig. 1), the blocking primer BivBlk and restriction enzyme BssSI as reported here have near universal application within the bivalves. The application of this basic approach could be extended to a wide diversity of consumers within the plankton community with minor sequence adjustments to the blocking primer. Although present in some planktonic consumers, the BssSI restriction site is not as ubiquitous outside of the bivalves. In these cases, it may be possible to identify an alternative restriction site or rely exclusively on a blocking primer. Slight variations to this basic dietary approach combined with next-generation sequencing capabilities will provide invaluable insights into the trophic ecology of small planktonic predators. Funding This work was supported by a Higher Education Authority of Ireland Strand III awarded to J.W.S. (grant number CRS/06/LYO1). Acknowledgements We would like to thank Sineád Gallagher for expert field assistance and insightful comments on early drafts of the manuscript and the staff of CAMBio for their laboratory support. References Altschul S. F. , Madden T. L. , Schaffer A. 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TI - Dietary analysis of small planktonic consumers: a case study with marine bivalve larvae
JF - Journal of Plankton Research
DO - 10.1093/plankt/fbt027
DA - 2013-07-01
UR - https://www.deepdyve.com/lp/oxford-university-press/dietary-analysis-of-small-planktonic-consumers-a-case-study-with-MqBWCpR8Ok
SP - 866
EP - 876
VL - 35
IS - 4
DP - DeepDyve
ER -