Ardura, A.

Garcia-Vazquez, E.

Samuiloviene, A.

Zaiko, Anastasija

OceanRep

Metabarcoding approach  in biodiversity and biosecurity surveys: a pilot study from the Baltic Sea Anastasija Zaiko,  Aurelija Samuiloviene, Alba Ardura, Eva Garcia-Vazquez  Email: anastasija@corpi.ku.lt          Klaipeda University                University of Oviedo Courtesy: JM Weslawski Challenges of marine surveys  Challenges of marine surveys Increasing human pressures, declining taxonomic expertise • Morphological identification methods are laborious • Require considerable taxonomic expertise • Often fail to identify cryptic species • Or species at the larval stage Photo credits: www.musselfree.org www.cefas.defra.gov.uk www.fao.org Crassostrea gigas Dreissena polymorpha Ostrea edule Risk to overlook or misidentify  non-indigenous, pathogen or indicator species Source: Valentini et al. 2009 Principle of the metabarcoding Zooplankton study in the Baltic Sea  Plume area (PA) “Sea gates” (SG) Klaipeda Strait (KS) •6 samples collected from ca. 2 m3 water each  •bulk DNA amplified with universal COI primers  •sequenced with a Genome Sequencer FLX (Roche)  •aligned against NCBI database  •compared to the morphologically analyzed samples Sequencing results • Approx. 100 000 good-quality sequences retrieved • About 75% of those resulted in positive alignment hits • 40 291 assigned (≥97% homology and >90% coverage) to 18 zooplankton taxa (species or genus level)  • Sequences assigned to zooplankton taxa  • Sequences discarded after filtering Species detected by metabarcoding (in red – those, detected exclusively by meabarcoding) Annelida Polychaeta Marenzelleria viridis Marenzelleria neglecta Crustacea Cladocera Bosmina coregoni Bosmina spp. Cercopagis pengoi Daphnia galeata Daphnia cucullata Evadne nordmanni Leptodora kindtii Pleopis polyphemoides Podon leukartii Copepoda Acartia tonsa Mesocyclops  leukarti Mollusca Bivalva Dreissena polymorpha Macoma balthica Mytilus sp. Gastropoda Hydrobia ulvae Rotifera Eurotatoria Keratella quadrata  Invasive species in the Baltic Annelida Polychaeta Marenzelleria viridis Marenzelleria neglecta Crustacea Cladocera Bosmina coregoni Bosmina spp. Cercopagis pengoi Daphnia galeata Daphnia cucullata Evadne nordmanni Leptodora kindtii Pleopis polyphemoides Podon leukartii Copepoda Acartia tonsa Mesocyclops  leukarti Mollusca Bivalva Dreissena polymorpha Macoma balthica Mytilus sp. Gastropoda Hydrobia ulvae Rotifera Eurotatoria Keratella quadrata  Considered to be absent from the study area Annelida Polychaeta Marenzelleria viridis Marenzelleria neglecta Crustacea Cladocera Bosmina coregoni Bosmina spp. Cercopagis pengoi Daphnia galeata Daphnia cucullata Evadne nordmanni Leptodora kindtii Pleopis polyphemoides Podon leukartii Copepoda Acartia tonsa Mesocyclops  leukarti Mollusca Bivalva Dreissena polymorpha Macoma balthica Mytilus sp. Gastropoda Hydrobia ulvae Rotifera Eurotatoria Keratella quadrata  ll ria viridis  >800 sequences found in all samples, aligned with high confi ence (>97% identity, >95% coverage) Traditional monitoring vs metabarcoding PERMANOVA results:  Factor  P____   Method  0,001 Location  0,017  Species detected by both techniques • Species detected only from morphological analysis  • Species detected only from sequencing  • Species shared between two approaches  Species detected by both techniques • Species detected only from morphological analysis  • Species detected only from sequencing  • Species shared between two approaches  BUT:   from species not identified with metabarcoding approach • 5 have no reference sequences in the public databases • others have reference sequences from specimens sampled elsewhere, not the Baltic Sea Biotic metrics and ecosystem health • Index calculated based on biomasses from morphological analysis  • Index calculated based on reads nr. from metabarcoding Biotic metrics and ecosystem health • Index calculated based on biomasses from morphological analysis  • Index calculated based on reads nr. from metabarcoding No statistically significant difference between 2 methods (Mann-Whitney test, p=0,07)  Potential application for other metrics: • % of copepod biomass • % of microphagous biomass • trend in  nr. of non-indigenous species Summing up… • Metabarcoding is a prospective tool for marine surveillance  • It does not require particular taxonomic expertise • It allows identification of cryptic life stages (eggs or larvae), detection of rare and sparsely distributed organisms  • It is applicable for early detection of environmental pests or indicator species  • Suitable for general biodiversity assessment and development of environmental quality metrics  BUT • Comprehensive reference databases needed • Quantification  should be elaborated • Marker validation / application of multiple markers Thank you! We thank Yaisel Borell for help with samples, Xavier Pochon and Susie Wood for critical comments on the abstract.   

Metabarcoding approach in biodiversity and biosecurity surveys: a pilot study from the Baltic Sea

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