The skin microbiome (based on 16S) of Antarctic Humpback whales (Megaptera novaeangliae)

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Bierlich K, Miller C, DeForce E, Friedlaender A, Johnston D, Apprill A (2019): The skin microbiome (based on 16S) of Antarctic Humpback whales (Megaptera novaeangliae). v1.1. SCAR - Microbial Antarctic Resource System. Dataset/Metadata. https://ipt.biodiversity.aq/resource?r=antactice_humpback_whale_skin_microbiome&v=1.1

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L’éditeur et détenteur des droits de cette ressource est SCAR - Microbial Antarctic Resource System. Ce travail est sous licence Creative Commons Attribution (CC-BY) 4.0.

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Cette ressource a été enregistrée sur le portail GBIF, et possède l'UUID GBIF suivante : 0144ad97-f933-4557-a599-0dd4c736066e.  SCAR - Microbial Antarctic Resource System publie cette ressource, et est enregistré dans le GBIF comme éditeur de données avec l'approbation du Scientific Committee on Antarctic Research.

Mots-clé

Metadata

Contacts

Couverture géographique

Healthy humpback whales in te Gerlache Strait, Palmer Deep Canyon and Bransfield Strait (Antarctica)

Couverture taxonomique

Bacteria and Archaea were targeted using universal primers for the 16S ssu rRNA gene (V4 region)

Couverture temporelle

Données sur le projet

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Méthodes d'échantillonnage

Skin samples were collected from the upper flank near the dorsal fin of humpback whales via standard biopsy techniques. Specifically, a 68-kg pull crossbow and floating Ceta-Dart darts with 40-mm surgical stainless steel tips collected a small skin and blubber sample roughly 5 mm wide and 20 to 30 mm deep, depending on the angle of the sampling dart on impact. Tips were sterilized using 8.25% concentrated bleach, allowed to dry, cleaned, and then doused in 100% ethyl alcohol before each use. The darts were retrieved by hand and the tips immediately placed into a sterile bag (such that the sample was not contaminated) and on ice for no more than 10 h before transfer to a cryovial via sterile tools for freezing at 80°C. The global positioning system (GPS) location and the regional names of the bay, strait, etc. were recorded for each skin sample collected. One skin sample, w132a, was collected from an acoustic tag placed on the animal.

Description des étapes de la méthode:

1. DNA was extracted from 1 to 30 mg of the top layer of the epidermis, with an average of 13 mg, from each sample using a DNeasy tissue kit (Qiagen, Valencia, CA, USA) and quantified using the Invitrogen Qubit 2.0 assay fluorometer (Life Technologies, Beverly, MA, USA). The V4 region of the SSU rRNA gene was amplified using barcoded primers (515F-Y, 5=-GTGYCAGCMGCCGCGGTAA-3=; and 806RB, 5=-GGACTACNVGGGTWTCTAAT-3=), broadly targeting bacteria and archaea. Samples and sterile water (negative control) were amplified in triplicate using PCR on a S1000 thermal cycler (Bio-Rad Laboratories, Hercules, CA, USA), as follows: 2 min at 95°C, followed by 30 to 35 cycles of 20 s at 95°C, 15 s at 55°C, and 5 min at 72°C, followed by 10 min at 72°C. Triplicate PCR mixtures contained 1 ul of DNA, 5 ul of GoTaq 5 Flexi buffer, 14.75 l of H2O, 2.5 l of a 25 nM MgCl2 solution, 200 nM each dinucleoside triphosphate (dNTP), and 0.25 l of a 5 units/ l GoTaq DNA polymerase solution per sample, along with 200 nM each primer. After PCR, amplification was assessed by mixing 5u l of each sample with 1 ul of 10,000 Sybr Safe dye run on a 1% agarose–Tris- borate-EDTA (agarose-TBE) gel. The replicate reaction mixtures were purified using Agencourt AMPure XP beads (Beckman Coulter, Inc., Pasadena, CA, USA) and quantified using the Qubit assay. Barcoded amplicons were sequenced using a 2x250-bp MiSeq Illumina format at the University of Illinois W. M. Keck Center for Comparative and Functional Genomics.

Citations bibliographiques

1. Bierlich, K. C., Miller, C., DeForce, E., Friedlaender, A. S., Johnston, D. W., & Apprill, A. (2018). Temporal and regional variability in the skin microbiome of humpback whales along the Western Antarctic Peninsula. Appl. Environ. Microbiol., 84(5), e02574-17. https://doi.org/10 .1128/AEM.02574-17

Métadonnées additionnelles