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Pipette, PCR, Paper: An update from the lab.

I am pleased to announce that we have some exciting findings from the blue sharks genetic study, which was undertaken in collaboration with Celtic Deep. The aim of this study was to see whether we could use a non-invasive method to collect DNA from free-swimming blue sharks for population studies. After collecting our genetic swabs from the sharks during the summer and early autumn, the rest of autumn and winter were spent in the labs at Newcastle University extracting the DNA, processing the samples, and analysing the results.

Blue shark in the Celtic Deep (Wales)

Throughout the summer field season, we were able to collect a total of 39 swab samples from what we recorded as 12 individual sharks by observation. For those of you who are unfamiliar with how we collected our samples we used a toothbrush attached to a pole to gently brush the skin of individuals as they swam past (See our 'Searching for blues' blog to find out more). This method was based on those used by Leiber et al., (2013) who carried out a similar study on Basking sharks, and Kashiwagi et al., (2015) who applied this method to Manta rays. Those of you who joined us this summer and saw the sampling first-hand will have seen that this method didn't disturb the sharks. Startle responses (quick short swim away before coming back) were the biggest reaction observed, and no shark left after being swabbed. In fact, all but one shark was sampled multiple times. We are delighted by the lack of disturbance this method had on the sharks, as this is a fundamental aspect of using non-invasive techniques. It is especially crucial when studying threatened and sensitive species and an essential factor when working with tour operators as causing displacement of the animals could impact their activities.

I spent many hours in the lab trying to find the best method to extract the genetic material from the toothbrushes, with the help and guidance of my supervisors at Newcastle University. Once we settled on a method and extracted the genetic material from all the toothbrushes, the endless pipetting began. This is where being slow and steady wins the race. Unfortunately, that is not my natural inclination, so I had to focus on slowing things down to avoid making mistakes. My biggest lesson with pipetting is not to become complacent. It is a repetitive process so easy to go into auto-mode, but you need to maintain concentration to avoid mistakes which could have (relatively) big consequences in terms of the amount of sample you have left or could result in needing to start a long process from the beginning.

I ran polymerase chain reactions (called PCRs for short) on the extracted genetic samples. You may have heard of PCRs as they are one method used to test for the COVID-19 virus. PCRs work by multiplying and making thousands of copies of pre-determined DNA regions depending on what you are looking for. We ran several PCR programs, adjusting the temperatures and cycle lengths for our samples to optimise the conditions for the DNA regions we were interested in. We had varying success depending on the program and which region of DNA we wanted to amplify. However, we were able to produce successful PCR products for all three DNA regions of interest. This included a gene known as CO1 which is commonly used to identify a species. We knew that we had swabbed blue sharks, however, there are lots of other organisms living on the skin of blue sharks and the sample could have been contaminated when passing through the water. So, by looking at the CO1 region we were able to identify which species the DNA came from if it wasn’t a blue shark. We did have several samples which in the results were identified as other species such as tunicates and ctenophores.

A key objective of this study was to determine the quality and quantity of DNA obtained from the skin swabs, to determine this we looked at regions of both the mitochondrial DNA and nuclear DNA as these two types can provide different information about an individual. We chose to look at the mitochondrial Control Region and nuclear ribosomal internal transcribed spacer two (ITS2) region. Looking at different regions of DNA allows us to begin to look at differences and similarities between individuals, including relatedness.

We are pleased to announce that our results add evidence for the use of non-invasive methods of collecting DNA from free-swimming sharks and rays, reducing the need to capture them. However, we encourage further work to determine the full potential and application of non-invasive methods to identify and track individuals in time and space to help inform fisheries management and conservation policies. These early findings are exciting, and with blue shark and other elasmobranch populations facing significant declines, developing genetic methods that prioritise the wellbeing of the animals and can be implemented on a large scale are essential.

Keep an eye out for publication with the full details of the study.

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