Our second day onboard was sunny and calm and so we took advantage and went sampling in the Minas Basin off the shore of Cape Blomidon. Many of the invertebrate species we that interest us in live inside sand or mud seabeds. So, to sample them we need to bring a chunk of the seabed up to the surface. To do this we use a grab sampler which, as the name suggests, grabs a piece of the sediment in its jaws. Working from the Polar Prince's small zodiac boats we deployed our 9x9 ponar mud grab. Sampling was tricky as parts of the seabed are rock here, and the grab sampler can't collect that. But, after a few attempts we collected four buckets of sand and pebble sediment. With several eager student helpers, we spent an enjoyable sunny afternoon ashore collecting at low tide. Flipping rocks to collect crabs and molluscs underneath them and hand sieving mud. Onboard we sieved the samples through a 0.5mm mesh to remove the animals from the sediment. Then under a microscope pulled out individual specimens. Most of the species we find are tiny polychaete worms, which often make up 60-70% of seabed samples.
Grab sampling from the zodiac. Credit: Students On Ice (SOI), Martin Lipman
Then began the work of curating the specimens for DNA barcoding. We gave each specimen a field identification and photographed it. Then we placed it into an individual vial of 95% ethanol (alcohol) for DNA preservation. We need to track the specimens carefully, so a sample ID label also goes into the jar with a unique number. We also must record collection information (e.g., region, date, habitat, GPS coordinates, depth, sampling protocol) for each specimen. This is critically important for the Barcode of Life project, both for quality control purposes and so that we can track exactly where species live, examine their ranges, and learn their habitats.
Christy pulling individuals of different species from samples. Credit: Students on Ice, Martin Lipman
A DNA barcode workflow and a BOLD specimen data page for a polychaete worm.
So, what is a barcode? DNA barcodes use a portion of a single gene – cytochrome c oxidase I – to identify the species to which an organism belongs. We remove a small piece of tissue from each specimen and extract the DNA from the cells present in it. Then we use primer to target the ‘barcode region’. The primers latch onto the DNA on either side of the target region, instructing where to make copies. Then we use PCR to make millions of copies of the gene region, so that we have enough to run though a sequencer. This gives us a 658 base pair nucleotide sequence, which is unique to each species. The reason scientists chose this mitochondrial gene region is because it does a great job of telling species apart. We don't find many differences between specimens from the same species (0-2%). But when we compare specimens from different species, we find differences of around 3-16%. These sequences are then visualized on a tree, which groups specimens based on how similar their barcodes are. We can use DNA barcoding to catalogue biodiversity, to assist with taxonomic identifications where morphological differences may be lacking (cryptic species), to identify various life stages and reproductive forms, incomplete specimens, or even market fish!
A tree built with DNA barcodes showing the distinction between species of polychaete worms collected from St. Andrews, NB
Our project will create a barcode library for Atlantic Canadian marine invertebrate species. It's funded by Fisheries and Oceans Canada under a stream which aims to develop tools for monitoring marine protected areas. Our project will link into the international Barcode of Life Project. Since it started in 2010, the Barcode of Life Project has grown to span 26 nations. The online workbench, Barcode of Life Datasystem (BOLD), now houses more than 9 million records representing 244 K animal species, 72 K plant species, and 24 K fungi and other species. I (Christy) was a part of the early days of the BOLD project, as a MSc student studying polychaete diversity in Canadian waters. Now, I am so happy to be a part of this initiative again. This will allow scientists to use new monitoring techniques like environmental DNA (eDNA) to study the ocean. Scientists collect eDNA samples, such as water samples, and extract DNA from them. They then match the DNA they find to barcode databases like BOLD. This is a non-invasive way of telling which species are or have been present in an area. But only if the barcode libraries are complete - that's where we come in.