Surveying estuarine meiofauna through DNA metabarcoding: optimization of sampling and molecular protocols

Maria FaisAB, Sofia DuarteA, Ronaldo SousaAC, Mehrdad HajibabaeiD, Carlos A. CanchayaB & Filipe O. CostaA

A Centre of Molecular and Environmental Biology, University of Minho, PortugalB Department of Biochemistry, Genetics and Immunology, University of Vigo, SpainC Interdisciplinary Centre of Marine and Environmental Research, University of Porto, PortugalD Centre for Biodiversity Genomics & Department of Integrative Biology, University of Guelph, Canada

ID 375

AbstractMeiofauna has important roles in the benthic domain, ensuring several ecosystem processes and services (1-2).To make possible the high-throughput profiling of this important community, a considerable improvement and optimization of metabarcoding protocols are stillrequired (3-4). To achieve this goal, in this study we tested:a) the amount of sediment necessary for eDNA extraction;b) the target genomic region and primer-pairs used for PCR amplification, and their impact on taxonomic profiling of estuarine meiofauna.

ResultsThe use of eDNA extracted from 10.0g of sample resulted in between14 and 230 more assigned OTUs compared to the second mostproductive amount (2.50g), for all except one primer-pair. 18S-V4 andCOI-A primers attained the highest number of OTUs assigned to taxa(>300 OTUs both), followed by V1-V2, V9 and COI-B (Fig.2A).Considerable differences were found in the taxonomic groups detectedamong markers and also among primer-pairs for each marker. Forexample, whereas 18S recovered the most of Ostracoda (Arthropoda),Rhabditophora (Platyhelminthes), Gymnolaemata (Bryozoa) and theGastrotricha Incertae Sedis, COI recovered all of the Demospongiae(Porifera), Stenolaemata (Bryozoa) and nearly all Malacostraca (Fig.2B).

eDNA was extracted from intertidal sediments (0.63, 2.50, and 10.0gsamples) collected in the Lima estuary, Portugal.Five PCR amplifications (Fig.1) were obtained by targeting 3hypervariable regions of the 18S rRNA gene (5-9), and two internalregions of the COI barcode (10-12).Amplicons were sequenced in an Illumina-MiSeq platform and theresulting forward and reverse reads were sorted-out using customizedprocedures in mothur (13-14).OTUs were clustered at 97% of similarity and subsequently BLASTedagainst customized databases from SILVA (18S rRNA) and BOLD (COI).

Material and Methods

Conclusions

• No single target genomic region or primer-pair captured comprehensively the phylogenetic diversity of the estuarine meiofaunal community.• Both amplification bias or taxonomic gaps in the reference libraries could explain the differences observed among loci and primers used.• For comprehensive community profiling at least two loci may be needed, using one or multiple primer-pairs for each one.• A considerable portion of the meiofauna taxonomic diversity can remain undetected if DNA is extracted from too small amounts of sediment.

Figure 1. Experimental design used for eDNA metabarcoding ofestuarine sediments collected in the Rio Lima estuary, Portugal.

A B

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References:

1. Wilson & Fleeger, Ch. 12 in Day et al. (Eds.), Est Eco II ed. (303-326), Wiley-Blackwell (2013)

2. Schratzberger & Ingels , J Exp Mar Biol Ecol in press (2017)

3. Cristescu, Trends Ecol Evol 29: 566-571 (2012)4. Creer et al. Methods Ecol Evol 7: 1008-

1018 (2016)5. Creer et al. Mol Ecol 19: 4-10 (2010)6. Stoeck et al. Mol Ecol 19: 21-31 (2010)

Acknowledgements:

This study has been funded by the project “The NextSea: Next generationmonitoring of coastal ecosystems in a scenario of global change” (operaçãoNORTE-01-0145-FEDER-000032), supported by Norte PortugalRegionalOperational Programme (NORTE 2020), under the PORTUGAL 2020Partnership Agreement, through the European Regional Development Fund(ERDF). MF and SD were supported respectively by a PhD(SFRH/BD/113547/2015) and a post-doc fellowship(SFRH/BPD/109842/2015) from FCT.

Figure 2. Proportion of OTUs of major taxonomic groups detected for each sediment amount and primer pair tested (A) and proportion ofOTUs of the most prominent Classes detected by each marker, primer pair and sediment amount (B):

Malacostraca (Arthropoda), Ostracoda (Arthropoda), Gastrotricha Inc.Sedis, Gymnolemata (Bryozoa), Stenolemata (Bryozoa),Demospongiae (Porifera), Hydrozoa (Cnidaria), Rhabditophora (Platyhelminthes)