Conceived and designed the experiments: CHF EK EMP JM. Performed the experiments: CHF EK EMP SH AG JM. Analyzed the data: CHF. Contributed reagents/materials/analysis tools: CHF SH AG JM. Wrote the paper: CHF EK EMP JM.
The authors have declared that no competing interests exist.
Molecular tools are now widely used to address crucial management and conservation questions. To date, dart biopsying has been the most commonly used method for collecting genetic data from cetaceans; however, this method has some drawbacks. Dart biopsying is considered inappropriate for young animals and has recently come under scrutiny from ethical boards, conservationists, and the general public. Thus, identifying alternative genetic collection techniques for cetaceans remains a priority, especially for internationally protected species.
In this study, we investigated whether blow-sampling, which involves collecting exhalations from the blowholes of cetaceans, could be developed as a new less invasive method for DNA collection. Our current methodology was developed using six bottlenose dolphins,
In this study, we show that blow-sampling provides a viable and less invasive method for collection of genetic data, even for small cetaceans. In contrast to dart biopsying, the advantage of this method is that it capitalizes on the natural breathing behaviour of dolphins and can be applied to even very young dolphins. Both biopsy and blow-sampling require close proximity of the boat, but blow-sampling can be achieved when dolphins voluntarily bow-ride and involves no harmful contact.
Information on kinship or relatedness is not only central to all theories of social evolution
In the last decade, a range of less invasive techniques, including skin swabbing
Blow-sampling was first attempted by Hogg
Blow is a relatively unexplored substance and thus its composition and full potential as a biological sample remains unexplored. Lung surfactant is likely to be the primary biological fluid in cetacean blow
In a pilot study, we first investigated whether or not blow samples could be collected from wild bottlenose dolphins in the eastern gulf of Shark Bay, Western Australia (25°47′S,113°43′E). Initially, we used a modified embroidery hoop with sterile filter paper stretched over its centre to collect the blow. We anticipated that absorbent filter paper would maximize sample collection. However, while this was the case, the filter paper inhibited successful DNA extraction from the sample. We did, however, successfully extract DNA from one individual, but were only able to amplify mitochondrial DNA. This preliminary work confirmed not only that blow could be successfully collected from small wild cetaceans, but that also these samples could be used to extract DNA information with appropriate methodological optimisation.
Our current methodology was developed using bottlenose dolphins,
A test tube was held inverted over the dolphin's blowhole and the dolphins were trained to exhale on cue (Figure 1). Four to six exhalations were collected per tube. We collected two sets of blow samples from each individual using sterile 50 ml polypropylene tubes (Fisherbrand) to test two different storage and transportation methods (detailed below). Both sets of blow samples were stored on dry ice for transportation to Georgetown University (Washington DC, USA). Once in the laboratory, the first set of blow samples were resuspended in 1 ml of 100% ethanol. The sides were scraped down using sterilized policemen, vortexed, and finally centrifuged at 3000 rpm for 3 minutes. Samples were then transferred to 1.5 ml microcentrifuge tubes to dry off the ethanol in a speed vacuum. The second set of blow samples were resuspended in 500 µl of TE buffer and centrifuged at 3000 rpm for 3 minutes. Excess TE buffer was carefully removed from all samples leaving a small amount at the bottom of the tube. DNA extraction of both sets of blow samples and blood samples were completed using a Qiagen DNeasy Blood and Tissue kit, using the animal tissues spin-column protocol. After DNA purification, blow samples were stored at −80°C until transportation to the University of Queensland (Brisbane, Australia) for DNA fingerprinting. A control sample of seawater was taken along with each blow sample set to assure that any DNA results were from blow samples and not seawater contamination.
For all six individuals, we found that their mitochondrial and microsatellite DNA profile taken from blow, matched their corresponding mitochondrial and microsatellite DNA profile collected from blood (see
To estimate whether the microsatellite profile collected from an individual's blow matched the microsatellite profile collected from its blood, we amplified 3 polymorphic dinucleotide microsatellite loci: Lobs_Di9 (black), Lobs-Di19 (red), and Lobs_Di21 (green)
To estimate whether the mitochondrial DNA profile collected from an individual's blow matched the mitochondrial DNA profile collected from its blood, we amplified a 426 base-pair fragment of the maternally inherited mitochondrial DNA control region using the primers dlp1.5
Overall, our study shows that DNA can be extracted from blow samples and that both mitochondrial and nuclear DNA can be successfully amplified using these samples. By comparing the DNA profile of blow to the DNA profile of its corresponding blood sample, we were able to demonstrate that DNA extracted from blow did not lead to problematic microsatellite amplification, such as non-specific amplification and allelic dropout
Microsatellite DNA profile of blow and blood from the remaining 5 individuals. The top 3 panels represent the three microsatellite loci amplified from DNA extracted from blow. The 3 lower panels represent the three microsatellite loci amplified from DNA extracted from blood. The microsatellite locus lobs_Di21 is coloured green. The microsatellite locus Lobs_Di9 is coloured black. And the microsatellite locus Lobs_Di9 is coloured red.
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Mitochondrial DNA profile of blow and blood from individuals 4 to 6. For ease of presentation we only show an 80 base pair long fragment (178 bp-255 bp) of the 434 base pair long sequence for three out of the six individuals. The remaining three DNA profiles are available on the online supporting material Figure S2. Additionally, the full sequences are available on GenBank accession numbers (HM581690-HM58701).
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We thank the marine mammal staff at National Aquarium: Deirdre Weadock, Shannon Daisey, Carolina Giraldo, Leah Whetzel, Justin Garner, Crystal Mumaw, Kerry Martens Tami, Goodman Denise, Davis Diane, Young April, Martin Jenna, and Giarrusso Ryan Ruegner. We also thank the animal health staff of the National Aquarium: Dr. Leigh Clayton, Dr. Kat Hadfield, Christine Steinert, Sarah McMillen, and Jill Arnold. In particular, we thank Valerie Lounsbury and Dr. Brent Whitaker.