IMIS

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.
Study Extent: Ninety-four skin samples were collected from 89 humpback whales in 12 different locations along the Western Antarctic Peninsula (WAP) during May and June 2010 (fall/late foraging season) and January and February 2013 (early summer/early foraging season). Five individuals were sampled twice during the study; three individuals were resampled on the same day, one a week later, and one was sampled in 2010 and 2013.
Method step description:

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.