Preliminary analysis of New Zealand scampi (Metanephrops challengeri) diet using metabarcoding
- Subject Areas
- Aquaculture, Fisheries and Fish Science, Bioinformatics, Genetics, Marine Biology, Molecular Biology
- Metanephrops challengeri, New Zealand scampi, Metabarcoding, PCR inhibition, Database analysis, Next generation sequencing, DNA polymerase, COI, 18S, Diet
- © 2018 van der Reis et al.
- This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, reproduction and adaptation in any medium and for any purpose provided that it is properly attributed. For attribution, the original author(s), title, publication source (PeerJ Preprints) and either DOI or URL of the article must be cited.
- Cite this article
- 2018. Preliminary analysis of New Zealand scampi (Metanephrops challengeri) diet using metabarcoding. PeerJ Preprints 6:e26667v2 https://doi.org/10.7287/peerj.preprints.26667v2
Deep sea lobsters are highly valued for seafood and provide the basis of important commercial fisheries in many parts of the world. Despite their economic significance, relatively little is known about their natural diets. Microscopic analyses of foregut content in some species have suffered from low taxonomic resolution, with many of the dietary items difficult to reliably identify as their tissue is easily digested. DNA metabarcoding has the potential to provide greater taxonomic resolution of the diet of the New Zealand scampi (Metanephrops challengeri) through the identification of gut contents, but a number of methodological concerns need to be overcome first to ensure optimum DNA metabarcoding results.
In this study, a range of methodological parameters were tested to determine the optimum protocols for DNA metabarcoding, and provide a first view of M. challengeri diet. Several PCR protocols were tested, using two universal primer pairs targeting the 18S rRNA and COI genes, on DNA extracted from both frozen and ethanol preserved samples for both foregut and hindgut digesta.
The selection of appropriate DNA polymerases, buffers and methods for reducing PCR inhibitors (including the use of BSA) were found to be critical. Amplification from frozen or ethanol preserved gut contents appeared similarly dependable, but metabarcoding outcomes indicated that the ethanol samples produced better results from the COI gene. The COI gene was found to be more effective than 18S rRNA gene for identifying large eukaryotic taxa from the digesta, however, it was less successfully amplified. The 18S rRNA gene was more easily amplified, but identified mostly smaller marine organisms such as plankton and parasites. This preliminary analysis of the diet of M. challengeri identified a range of species (13,541 reads identified as diet), which included the ghost shark (Hydrolagus novaezealandiae), silver warehou (Seriolella punctate), tall sea pen (Funiculina quadrangularis) and the salp (Ihlea racovitza), suggesting that they have a varied diet, with a high reliance on scavenging a diverse range of pelagic and benthic species from the seafloor.
Updated version as per reviewers suggestions and feedback.
PCR profile designed for the optimum DNA amplification using either the COI or 18S primers
(A) Initial denaturation at 95 °C for 60 seconds. (B) 20 “touch-up” cycles, with each cycle having a denaturation step at 95 °C for 30 seconds, an annealing step starting at 45 °C for 30 seconds (each annealing cycle increases 1 °C every cycle) and a final extension step at 72 °C for 30 seconds. (C) 20 “touch-down” cycles, with each cycle having a denaturation step at 95 °C for 30 seconds, an annealing step starting at 65 °C for 30 seconds (each annealing cycle decreases 1 °C every cycle) and a final extension step at 72 °C for 30 seconds. (D) 10 cycles, with each cycle having a denaturation step at 95 °C for 15 seconds, an annealing step at 45 °C for 20 seconds and a final extension step at 72 °C for 30 seconds. (E) Final extension cycle at 72 °C for 60 seconds and holding at 15 °C.
Platinum Taq reaction
Reagent volumes and concentrations used in a 25 μl Platinum Taq reaction.
Reagent volumes and concentrations used in a 25 μl Bioline reaction.
Processed reads for COI and 18S
Resulting totals for paired, merged and cleaned reads for COI and 18S.
The DNA concentration and purity ratios per individual for each digesta source
Nucleic acids have strong absorbance at 260 nm, which is also the wavelength where purines and pyrimidines peak. At 280 nm, proteins and phenolic compounds have a strong absorbance. A wavelength of 230 nm is known to absorb organic compounds. Pure DNA should ideally be 1.8 for A260/A280 and close to 2.0 for A260/A230 (Watts 2014).
COI diet reads
COI genus reads were filtered from the COI cleaned reads and then categorized into DNA negative reads, lobster and/or terrestrial reads and diet reads.
18S diet reads
18S genus reads were filtered from the 18S cleaned reads and then categorized into DNA negative reads, lobster and/or terrestrial reads and diet reads.
Taxa identified as diet taxa (genus and/or species level) from the COI databases
The taxa are separated into their OTUs with their assigned taxonomic identity, hit counts, Midori RDP confidence levels, NCBI e-values and grouping.
COI diet sequences
COI OTUs and assigned sequences (genus and/or species level).
Taxa identified as diet taxa (genus and/or species level) from the 18S databases
The taxa are separated into their OTUs with their assigned taxonomic identity, hit counts, SILVA RDP confidence levels, PR2 RDP confidence levels, NCBI e-values and grouping.
18S diet sequences
18S OTUs and assigned sequences (genus and/or species level).