Aquatic Barcoding

There isn't a shopper alive who doesn't know what a barcode looks like.  We see them on virtually everything we buy – labels with distinct series of vertical bars and white spaces of varying widths.  The labels are read with a scanner, which measures reflected light and translates the coding into numbers and letters that are passed on to a computer.  Barcodes track just about everything there is to know about a product and its status in the supply chain. 

But it is not just in the world of commerce that barcoding shows up.  It is also employed in the world of aquatic ecology where barcodes are used in the genetic identification of fish, invertebrates, and even parasites.  This genetic approach to species identification is very useful for addressing a whole host of questions, including ones relevant to resource and conservation management, as it can be more sensitive, more accurate, and more time- and cost-effective than traditional observational methods of identifying species from close relatives.    That's because visual identification requires intact specimens, the presence of morphological characteristics that can separate species (which are lacking in some groups), and specialized expertise to conduct the labour-intensive examination of each specimen.

Dr. Abbott (right) and post-docs Dr. Magalie Castelin (middle) and Dr. Niels Van Steenkiste (left) collecting specimens for the DNA barcode library and associated biological specimen collection. These specimens and associated data become a permanent genetic resource to aid in invasive species identification and detection into the future.

Dr. Cathryn Abbott, a research scientist working out of DFO's Pacific Biological Station (PBS) in Nanaimo, BC puts her expertise in ecological and evolutionary genetics to good use in the diagnostics of aquatic animal pathogens and in the early detection of aquatic invasive species (AIS).  Barcoding is a critical element in her work. 

The introduction of invasive species into native habitats not only can be ecologically disastrous, but economically as well, costing the Canadian economy tens of billions of dollars annually.  One federal government response to this problem is interdepartmental research in genomics on the "Protection of Canadian bio-diversity and trade from the impacts of global change through improved ability to monitor invasive alien and quarantine species."  The overarching aim of the project is to enable early detection of invasive species to protect Canadian natural capital and the economy.  The work falls within the mandate of the Government of Canada's Genomics R&D Initiative (GRDI), which coordinates federal science departments and agencies in the field of genomics research.

There are a number of areas of study associated with this research, including a sub-project called Barcoding of aquatic invasive species of highest risk to Canadian native fauna and trade.  Dr. Abbott oversees this sub-project, which is divided into three parts.  One part is focused on freshwater finfish.  Here researchers are looking to early detection of AIS that can result from the importation of live fish through the live food and aquarium trades, both of which are potential pathways of AIS introduction.  Dr. Abbott's DFO colleague Nellie Gagné in Moncton, New Brunswick is handling the finfish side of the work.  

Another part of the project focuses on invertebrate AIS, whose pathway of introduction is usually through commercial ships via their ballast tanks or through hull fouling on recreational boats.  Dr. Abbott is investigating these.  

Environment Canada is focusing on a third area - aquatic parasites, which can be introduced through multiple pathways. 

Students and post-docs work together in Dr. Abbott’s lab at the Pacific Biological Station, shown here extracting DNA for barcoding.

Barcoding projects have four components: the retrieval of specimens, identification by visual inspection, DNA sequencing (i.e. 'DNA barcoding'), and the entry of all data associated with each specimen into a reference databaseFootnote 1. These databases are ultimately used to assign unknown specimens to known species, based on matching their DNA sequence to a reference record in the database.

The goal of DNA barcoding is to use a short segment of DNA sequenceFootnote 2 from a standardized genetic regionFootnote 3 to identify what species is present.  Dr. Abbott: "DNA sequences are useful for identifying the species of unknown samples. The whole point of an interdepartmental project like this is to develop a comprehensive sequence library. The database has to be able to substantiate the link between genetic sequences and taxonomic identification.  To increase our confidence in species identification we are building a database that has DNA sequences from multiple genetic regions."

A lot of effort is being focused on generating reference DNA sequence databases for native Canadian fauna that are within taxonomic groups that have high profile invaders.  That way researchers can reliably distinguish an invasive species from a native species.  Such reliability is very important because this method can be used for regulatory purposes.

In the case of freshwater finfish, samples are obtained through a mix of material from the Royal Ontario Museum and from freshly collected specimens from DFO or other sources. 

An invasive on an invasive. Collected from Boundary Bay, British Columbia: the introduced slipper limpet Crepidula convexa (native to the east coast of North and central America) on the invasive Japanese oyster drill Ocenebra inornata (left and right in photo); and on the Japanese mudsnail Batillaria attramentaria (middle). Photo: Rick Harbo (retired, DFO) Research Associate, Royal BC museum.

With aquatic invertebrates most of the material they have sequenced so far comes from freshly collected specimens, through various collaborations or DFO field programs.  Dr. Abbott: "When a fresh specimen comes in, a genetic sample and a morphological (form and structure) voucher need to be prepared for the DNA barcode archived collection.  It needs to be taken and preserved for genetic analysis.  That involves taking a tissue sample for immediate DNA extraction and sequencing as well as retaining some tissue pieces for archival purposes.  This is so that, if needed, they can be used later to do any extra analyses or to repeat the work for verification purposes."

The morphological voucher also requires appropriate preservation.  This is a specimen that can be gone back to so that the specimen's taxonomy can be cross-checked.  All of the metadata around each specimen needs to be entered into the database.  A tissue voucher is also prepared.  A tissue voucher is subsampled tissue preserved to keep its molecular properties intact for future analysis.  And of course a DNA sequence is generated.

When it comes to analysis Dr. Abbott notes that "An important piece of generating a valid DNA barcoding database is to ensure that the entries are correctly identified.  Ultimately, the goal is to resolve cases where there is conflict between the DNA-based taxonomy and the morphological taxonomy. In short, the signature in the genes has to match the taxonomic classification for barcoding to work."

"And that," adds Dr. Abbott, "requires an integrated approach to taxonomy where a combination of genetic, morphological, genealogical, and ecological data are collected to define species units.  This is a very effort-intensive element of building a validated DNA barcoding database because multiple lines of evidence are required."

She adds that this is not a simple task since AIS are taxonomically very diverse.  The work involves a very broad range of species and they need to make sure that any method they develop and implement is reliable, because there can be a high cost to both false positives and false negatives.  And as she emphasizes, "High confidence is needed before we can reliably use it as the basis for a DNA-based detection tool."

It is clear why the Government of Canada sees this work as vital to the process of fighting off the scourge of AIS.  Once AIS establish themselves and spread in their non-native environment they are virtually impossible to eradicate. The best medicine is the earliest detection possible which gives you a fighting chance of preventing invasive populations from becoming established.  And if populations do become established, genetic tools can be used to monitor and mitigate their spread.  DNA barcoding can serve a dual purpose: it can play a role in resolving taxonomic uncertainties as part of an integrated taxonomic approach, and can serve as an innovative device for non-experts who need to make a quick identification of AIS. 

Prevention is clearly the best cure, and the entry of the barcoders into the process is essential to doing exactly that.

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