of three-spined stickleback in a coastal ecosystem Jakubaviit E* 1 , - - PowerPoint PPT Presentation

DNA metabarcoding reveals diverse but selective diet

• f three-spined stickleback in a coastal ecosystem

Jakubavičiūtė E*1, Bergström U2, Haenel Q3, Bourlat SJ4, Eklöf J5

1 Nature Research Centre, Vilnius, Lithuania 2 Department of Aquatic Resources, Institute of Coastal Research, Swedish University of Agricultural Sciences (SLU), Öregrund, Sweden 3 Zoological Institute, University of Basel, Switzerland 4 Department of Marine Sciences, University of Gothenburg, Box 463, 405 30 Gothenburg, Sweden 5 Department of Ecology, Environment and Plant Sciences, Stockholm University, Sweden

Tallinn, 2017

Outline

• Introduction: DNA metabarcoding
• Stickleback diet diversity
• Stickleback diet selectivity
• Comparing the methods: visual stomach content analysis vs DNA

metabarcoding

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DNA metabarcoding

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Kress et al., 2015

• DNA barcode - any DNA sequence used for identification at any

taxonomic level;

• DNA barcoding - taxon identification using a standardized DNA

region;

• DNA metabarcoding - the use of NGS to identify multiple

species in a sample using DNA barcodes;

• OTU – operational taxonomic unit - gene sequence reads,

clustered based on sequence similarity.

DNA metabarcoding

• Biodiversity assesment, trophic interactions, community composition...
• Environmental DNA (eDNA) metabarcoding
• eDNA in aquatic environments - non-invasive monitoring tool

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Material & Methods

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• 196 three-spined stickleback samples

TL=57.7 ±7.6 mm (SD)

• Visual inspection of stomach

contents

• DNA metabarcoding
• Zooplankton abundance data in bays
• Benthos abundance data in bays

Workflow

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Fish dissection, visual identification of stomach contents DNA extraction PCR

• Amplification of 313 bp region

in COI (Leray et al., 2013)

• Universal and blocking primers

Sequencing

• Illumina MiSeq platform, final

dataset of 10 586 546 reads

Taxonomic assignement

• Sequences clustered into OTUs

and compared against reference databases

Taxa1 (OTU).... # of reads Taxa2 (OTU).... # of reads

Data analysis

• %Nbar - proportions of prey in the stomachs based on the number of OTU reads

per taxon

• %Fvis and %Fbar - frequency of occurrence based on barcoding and visual data
• Jacobs’s index for diet selectivity estimations
• J=(r−p)/(r+p−2pr)
• r - %OTU reads of certain prey species in stomach (%Nbar)
• p - proportion of certain prey in the environment (zooplankton/ benthos)

J [-1;1]

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Diet diversity of three-spined stickleback

• 15 phyla
• 27 classes
• 52 orders
• 66 families
• 83 genus
• 84 species
• Primary prey – 103 taxa

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• 120 taxa revealed by metabarcoding in the stomachs of sticklebacks

Proportion of different classes in stomachs (%Nbar)

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Insecta 48% Maxillopoda 19% Branchiopoda 15% Ostracoda 10% Malacostraca 3% Actinopterygii 2% Bivalvia 1% Gastropoda 1% Polychaeta 1%

Chironomidae 55% Podonidae 19% Tachidiidae 13% Temoridae 6% Cyclopidae 2% Gasterosteidae 2% Asellidae 1% Tellinidae 1% Cytherideidae 1%

Proportion of different families in stomachs (%Nbar)

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Main prey species

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Tanytarsus usmaensis Pleopis polyphemoides Tachidius discipes Eurytemora affinis Chironomus aprilinus Dicrotendipes modestus 5 10 15 20 25 30 35 40 10 20 30 40 50 60 70 80 90 100 Relative abundance, % Nbar Frequency of occurence, %Fbar

Diet selectivity

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preferred rejected *P<0.05 **P<0.01

Fish size and diet

Medium Large P values (4.5 – 6.5 cm) (>6.5 cm) Small (<4.5cm) 0,0037 0,0062 Large (>6.5 cm) 0,6576 R values Small (<4.5cm) 0,2623 0,2821 Large (>6.5 cm)

• 0,02179

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• Differences in stomach content depending on fish size

(Permanova, P=0.044).

• Diet of the smallest fish (<4.5 cm) group differed from the
• thers

Table 1. Anosim results, differences in diet between fish size groups.

20 40 60 80 100 <4,5cm 4,5-6,5 cm >6,5 cm S M L % Nbar Diptera Harpacticoida Calanoida Cyclopoida Diplostraca Podocopida Amphipoda Isopoda Littorinimorpha

n=8 n=158 n=30

Mean taxonomic rank assigned to items within individual stomachs

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Number of taxa identified per stomach

DNA metabarcoding vs visual inspection

Compliance of the two methods: visual vs barcoding

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Annelida Insecta Chironomidae Branchiopoda Maxillipoda Malacostraca Amphipoda Ostracoda Bivalvia Gastropoda R² = 0.8363 10 20 30 40 50 60 10 20 30 40 50 60 70

Frequency of occurence, % Fvis Relative abundance, % Nbar

DNA metabarcoding vs visual inspection

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Visual inspection DNA metabarcoding Mean taxonomic rank Order Genus Mean number of taxa identified per stomach 1.96 ± 1 (SD) 21.7 ± 8.8 (SD) Total number of taxa identified 21 120

Methodological shortcomings

• Secondary consumption
• 103 out of 120 taxa are primary prey?
• Some taxa detected by visual inspection only (Temora longicornis, Bosminidae, Hydracarina),

some may be underestimated (Bivalvia)

• Quantification
• primer-template mismatches
• Species differ in amount of DNA/per gram
• taxon-specific differences in how well DNA amplifies
• PCR bias in amplification may result in skewed recovery of sequences

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Take home message

• In general, the two methods gave consistent results with the same

prey taxa dominating.

• High-throughput

DNA sequencing is a promising method for estimating the composition and richness of three-spined stickleback diet, but that some further technical development is needed to efficiently capture all species in the diet.

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Aitäh!

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Eglė Jakubavičiūtė PhD student ejakubaviciute@ekoi.lt, ejakubaviciute@yahoo.com Laboratory of Marine Ecology Nature Research Centre, Akademijos str. 2, LT-08412, Lithuania