FURTHER FACTS FROM FAECES:Dietary DNA Barcoding Using High Throughput Sequencing Bruce Deagle and Simon Jarman
Why study animal diet?“Statistics show that of those who contract the habit of eating, very few survive.” George Bernard Shaw Decline of Steller sea lions From Trites and Donnelly (2003) Mammal Review 33:3-28
Baleen Flighted whales birds PenguinsSeals Toothed whales Demersal fish Pelagic fish Squid Amphipods Krill Salps Copepods Unicellular Organisms
1. Observe feeding 3. Collect gut contents 4. Collect faeces 2. Tissue sample identify hard-parts ‘the one-eyed man is king in the land of the blind’ Different approaches to studying diet
Captive Feeding Trial Steller sea lions (Eumetopias jubatus)• Reliability of prey DNA recovery from faeces?• Persistence of genetic signal?• Quantitative estimates of diet?• Quality of prey DNA?
Feeding Trial - Methods Surf Smelt Squid(Hypomesus pretiosus) (Loligo sp.) Pulse prey itemsSockeye Salmon Pacific Herring(Oncorhynchus nerka ) (Clupea pallasii ) Group Specific PCR - DNA was extracted from soft material (n=108) - Sea lion DNA was dominant component Jarman et al. (2004) Molecular Ecology 13:1313-1322
Feeding Trial - Results Frequency of detection • Reliable prey DNA detection for basic dietary items • Each species has a equal chance % prey positives (n=108) of being detected, squid (6% of diet) is consistently detectedSquid 94% • DNA from pulse prey items turnedHerring 94% up in faeces produced between 12 and 48 hours after ingestionSmelt 92%Salmon 87%Mean 92% Deagle et al. (2005) Molecular Ecology 14:1831-1842
Quantitative Estimates Do proportions of DNA in faeces reflect intake? Proportion of DNA in Faeces (qPCR) • Prey DNA in faeces from 10 diets fed to Steller sea lions. 4 Diet Items Herring Eulachon Squid Rockfish Proportion in diet by mass Bowles et al. (2011) Molecular Ecology Resources 11: 530-540Deagle and Tollit (2007) Conservation Genetics 8: 743-747
Prey DNA Quality Herring DNA- Scat 1 25000 - Mini-barcodes best 20000 (< 200 bp)Copy number Copy Number 15000 10000 5000 0 0 50 100 150 200 250 300 PCR product size PCR product size Deagle et al. (2006) Frontiers in Zoology e3:11
Prey DNA Quality Herring DNA- Scat 1 25000 F(x)= αe-λ x - Allows quantification of 20000 DNA damageCopy number λ = probability of a break Copy Number 15000 10000 5000 0 0 50 100 150 200 250 300 PCR product size PCR product size Deagle et al. (2006) Frontiers in Zoology e3:11
Passmore et al. (2006) Marine Biotechnology Casper et al. (2007) Marine Biology Dunshea (2009) PLoS ONE Deagle et al. (2007) PLoS ONE
Next generation sequencingMarguilies et al. 15 September 2005: Volume 437: 376-380
Australian fur seal(Arctocephalus pusillus doriferus)
Fur Seal Diet Questions• Benthic foragers, pelagic prey?• Are more large commercially important fish consumed than has been estimated?• Are sharks and/or rays consumed?
DNA extraction from individual faecal samples PCR 1* PCR 2* PCR 3 PCR 4 Chordata Chordata/Cephalopoda Cephalopod Bilateria mtDNA 16S mtDNA 16S nuclear 28S nuclear 18S*With blocking primer *With blocking primer Pool PCR amplicons Pyrosequencing Roche GS FLX Bioinformatic sorting of sequences Prey species ID
Choice of Genetic Markers MtDNA COI(protein coding gene) Sequence position (bp) MtDNA 16S 1. mtDNA 2. mtDNA(ribosomal DNA gene) short long Based on alignment of DNA sequences from 100 teleost fish (data from Miya et al. 2003, Molecular Phylogenetics and Evolution 26: 121-138)
Methods- PCR blocking mtDNA Bony fish 1 short Bony fish 2mtDNA long Bony fish 3 Fur seal Blocking Oligo Shark Squid 0.05
Methods- Melting curve analysis of PCR PCR without blocker PCR with blocker Fur seal PCR products∆ fluorescence dF/d(T) ∆ fluorescence dF/d(T) Fish PCR products Temperature Temperature Figure: Melting profiles of PCR products from six fur seal faecal DNA extracts. Vestheim et al. (2011) Methods in Molecular Biology 687:265-274
Results- Comparison of mtDNA primer sets Marker Short Primer Set ~150 bp mtDNA 16S n= 10585 Seal Rocks Jack Mackerel (Trachurus sp.) n= 2959 RedbaitLocation Lady Julia Percy (Emmelichthys nitidus) Blue Mackerel (Scomber australasicus) Barracouta n= 2990 (Thyrsites atun) Other Fish The Skerries n= 4636 Deagle et al. (2009) Molecular Ecology 18:2022-2038
Results- Comparison of mtDNA primer sets Marker Short Primer Set Long Primer Set ~150 bp mtDNA 16S ~300 bp mtDNA 16S n= 10585 n= 2102 Seal Rocks Jack Mackerel (Trachurus sp.) n= 2959 n= 524 RedbaitLocation Lady Julia Percy (Emmelichthys nitidus) Blue Mackerel (Scomber australasicus) Barracouta n= 2990 n= 908 (Thyrsites atun) Other Fish The Skerries n= 4636 n= 670 Deagle et al. (2009) Molecular Ecology 18:2022-2038
One-eyed king?Who is eating what: diet assessment using Next Generation Sequencing Pompanon et al. (In Press) Molecular Ecology
Nick Gales Paige Eveson Acknowledgements Roger Kirkwood Mark Hindell Ella Bowles Andrew Trites Nuka, Hazy et al. Dom Tollit Bob Ward
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