DNA metabarcoding of the prey and microbiome of museum specimen Antarctic trematomid fishes
Citation
Heindler F, Christiansen H, Frédérich B, Dettaï A, Lepoint G, Van de Putte A, Volckaert F (2020): DNA metabarcoding of the prey and microbiome of museum specimen Antarctic trematomid fishes. v1.3. SCAR - Microbial Antarctic Resource System. Dataset/Occurrence. https://ipt.biodiversity.aq/resource?r=historic_antarctic_fish_dataset_2019&v=1.3 https://doi.org/10.15468/5axtcc accessed via GBIF.org on 2024-09-01.Description
In this dataset, stomachs and hindguts were sampled from 225 Trematomus specimens from the Natural History Museum London. Fish specimen were collected between 20 and 100 years ago and fixed in either formaldehyde or ethanol. A 313 bp fragment of the cytochrome c oxidase subunit I (COI) was amplified and sequenced for prey item identification in the stomach and a 450 bp region of the 16S rRNA gene to investigate microbiome composition in the gut system.Sampling Description
Study Extent
Stomach and hindgut samples of 225 specimens of the genus Trematomus were obtained from the Natural History Museum, London. Sampling dates ranged from 1901 to 1988, sampling locations were in the Southern Ocean around the Antarctic continent. Contemporary samples were caught with hook and line in the vicinity of the Gerlache Strait, Antarctic Peninsula in the season of 2017–2018.Sampling
Fish were carefully dissected: stomachs were opened to remove stomach content and a small portion of the hindgut (1 cm) was removed. Stomach content and hindgut were stored separately in 70% ethanol.Quality Control
During molecular laboratory work special care was taken to prevent (cross-) contamination of samples. Workbench wipes (workbench contamination), human saliva wipes (human contamination) and no-template extractions (blanks) were included as contamination controls for amplification and sequencing.Method steps
- A large piece of stomach content (0.5 × 0.5 cm) or the entire piece of hindgut (1 cm) was placed into screwcap microtubes with 500 μl of Phosphate Buffered Saline (PBS) at pH 9. Tissue was fragmented thoroughly in each tube to ameliorate efficiency. Samples were heated to 100°C for 10 min, left to cool on ice for 5 min and then spun down with 20,000 × g for 5 min. PBS was carefully removed without taking any tissue and replaced by 500 μl of PBS at pH 7.2 and again heated to 100°C for 10 min. PBS was again carefully removed and further purification steps were conducted using the commercial Nucleospin® Tissue (Macherey-Nagel, Accession number: 740952) DNA extraction kit following the manufacturer's protocol.
- For prey identification a 313 bp region of the COI gene was amplified from the stomach content using the tailed primers NGSmlCOIint and NGSjgHCO2198. The V3 and V4 region (460 bp) of the 16S rRNA gene was amplified using the tailed primers 16s-IllumTS-F and 16s-IllumTS-R to assess the microbiome composition. The reaction mix for the amplicon PCR for COI contained 12.5 μl of MytaqTM 2x Mix (Bioline, Accession number: BIO-25041), 0.5 μl of each primer (20 μM), 10.5 μl of molecular grade water and 1 μl of DNA template with a PCR profile of 10 s of denaturation at 95°C, 30 s of annealing at 62°C and 60 s elongation at 72°C for 16 cycles with the annealing temperature dropping every cycle by 1°C, followed by 25 cycles with an annealing temperature at 46°C. The reaction mix for the amplicon PCR for 16S contained 12.5 μl of MytaqTM 2x Mix, 2.5 μl of each primer (1 μM), 2.5 μl of DNA template (5 ng ul−1) and 5 μl of molecular grade water with a PCR profile of 60 s of initial denaturation at 95°C followed by 25 cycles of 15 s denaturation at 95°C, 15 s of annealing at 55°C and 10 s elongation at 72°C, finishing with a final extension of 72°C for 300 s. PCR products were cleaned up using Agencourt AMPure XP beads (Beckman Coulter, Accession number: A63882) following the manufacturer's instructions with a bead to template ratio of 0.8 to 1. Thereafter followed an indexing PCR, which binds a unique primer barcode to each respective sample following Lange et al. (2014) with a PCR mix of 10 μl of MytaqTM 2x Mix, 0.5 μl of each forward and reverse indexing-primer (to form a unique identifiable primer combination for each sample; 20 μM) and 9 μl of DNA template with a PCR profile of an initial denaturation of 1 min at 95°C followed by 15 cycles of denaturation for 15 s at 95°C, 15 s of annealing at 51°C and 10 s of extension at 72°C finishing with a final extension of 5 min at 72°C. The PCR product was cleaned up again, then quantified using the commercial Quant-iTTM Picogreen® kit (Thermo Fisher) and pooled, if sufficient template (20 ng) was available. Sequencing took place on an Illumina MiSeq PE 3000 (Genomics Core, KU Leuven, Belgium).
Taxonomic Coverages
Fish specimen of the genus Trematomus, with DNA samples of the stomach and gut for prey composition and micro biome.
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Trematomus eulepidotusrank: species
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Trematomus penelliirank: species
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Trematomus hansonirank: species
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Trematomus eulepidotusrank: species
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Trematomus brachysomarank: species
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Trematomus bernacchiirank: species
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Trematomus borchgrevinkirank: species
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Trematomus newnesirank: species
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Trematomus loennbergiirank: species
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Trematomus scottirank: species
Bacterial and Archaeal microbiome in the stomach and gut of the fish was profiled with the 16S ssu rRNA gene
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Bacteriacommon name: Bacteria rank: domain
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Archaeacommon name: Archaea rank: domain
Prey (eukaryotes) of the fish were investigated with the COI marker gene
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Eukaryotacommon name: Eukaryotes rank: domain
Geographic Coverages
Various places in the Souther Ocean and the coastal waters of the Antarctic continent. Geographic coordinates not available for all the museum specimen.
Bibliographic Citations
- Heindler F.M., Christiansen H., Frédérich B., et al. (2018) ‘Historical DNA Metabarcoding of the Prey and Microbiome of Trematomid Fishes Using Museum Samples’. Frontiers in Ecology and Evolution 6: 151, https://doi.org/10.3389/fevo.2018.00151. - https://doi.org/10.3389/fevo.2018.00151.
Contacts
Franz Heindleroriginator
University of Leuven
Leuven
BE
Henrik Christiansen
originator
University of Leuven
Leuven
BE
Bruno Frédérich
originator
University of Liège
Liège
BE
Agnes Dettaï
originator
Muséum National d'Histoire Naturelle
Paris
FR
Gilles Lepoint
originator
University of Liège
University of Liège
BE
Anton Van de Putte
originator
University of LeuvenRoyal Belgian Institute of Natural Sciences
Brussels
BE
Filip Volckaert
originator
University of Leuven
Leuven
BE
Heindler Franz
metadata author
University of Leuven
Leuven
BE
Maxime Sweetlove
metadata author
Royal Belgian Institute of Natural Sciences
Brussels
BE
Heindler Franz
content provider
University of Leuven
Leuven
BE
Franz Heindler
administrative point of contact
University of Leuven
Leuven
BE