04 July 2014

Finding Nemo with DNA barcoding



This blog post was written by postgraduate student Jonathan Ridden as part of the course, Research Methods in Ecology (Ecol608). Jonathan revisits a Lincoln University research area that looks at using DNA to identify invasive species (fish in this case) published in 2012 and 2013.

I remember a pond back in my hometown of Timaru that was located in Centennial Park. It was a really neat place to go as a child, somewhere you could run rampant. While the pond was not swimmable due to ‘duck itch’, (which relates to nasty little parasites that parasitise ducks and cause allergic reactions!), it was somewhat liveable for other biota, and you could still skim stones over it. It was a place where I had my first kayak lesson (I think my school didn’t have a very high regard for student health now that I look back on it). However, when I was about 12 or 13, the pond’s health deteriorated rapidly. The main cause of this was attributed to the arrival of a new species of fish into the pond: the koi carp.

These carp were accidentally introduced to New Zealand in the 1960's as part of a goldfish consignment from Asia and Europe (the carp’s native continents). The multiple impacts these fish have on waterways in general is very bad. They stir up the pond bottom, muddying the water and destroying other aquatic organisms’ habitat. Of course, this leads to further degradation of water quality and eventual decline of associated waterfowl populations. On a good day in Centennial Park, you can still find ducks, but much fewer than when I was young, to feed some bread. This example just goes to show the impact something like an exotic and highly invasive fish species can have on waterways in New Zealand.

Group of ducks floating on a pond. Now you can only imagine whats going beneath the surface! Fancy a swim? Photo by Thom Quine


New Zealand has a reputation for having a very strict and stern view on biosecurity and bio-protection.  The impacts of invasive species to New Zealand ecosystems can have detrimental impacts on the native flora and fauna. Fish are a major threat, as there are other species that are just as invasive as the koi carp (e.g. perch, rudd and catfish). One potential avenue for invasion of fish species into New Zealand is via the ornamental aquaculture trade. When bringing fish into New Zealand for sale and commercial ventures, many different types of fish with highly variable morphology (fish with different colours, sizes and shapes) cross our borders. 



These two fish look similar, but are different
 species based on DNA analysis.
Species A is Puntius filamentosus,while
 species B is P.assimilis.
(Adapted from Collins et al 2012)
A major issue of the industry is a lack of confidence in the identity of the fish species, as people who sell these fish are usually not trained in fish identification. This produces a major threat to New Zealand aquatic environments because, if a fish that is a potential high-impact invasive species is misidentified, it could get pass the border and into our natural environment. Undesired fish species can bring along bacterial pathogens which, if exposed to native fisheries and aquatic environments, would damage the country’s fish populations. That includes trout populations, which recreational anglers would not be happy about! So the question at hand is: how can incoming fish be identified efficiently when they can all look very similar or have never been seen here before?
 
Many different methods can be used to distinguish the species of fish. This includes morphological identification of specimens using keys. However, this process can be difficult and requires highly trained individuals to work quickly and efficiently at the border to identify risk species. An alternative, rapidly improving method to identify organisms is to use DNA barcoding. This is done by taking a sample of DNA from a specimen to the laboratory and using molecular methods, producing a unique barcode attributed to that specimen. Water in which fish has been living in during transport between countries, can be used to get DNA sequences from, rather than clipping a part of the fish off (See this paper). Rupert Collins and colleagues from Lincoln University completed a study which used a suite of techniques centered on DNA barcoding, in order to accurately identify incoming ornamental cyprinid fish species at the border. This involved developing a reference library of fish specimens and their associated DNA sequences, using the common mitochondrial barcoding gene region cytochrome c oxidase subunit I (or CO1 for short). There are several benefits to producing a physical library of specimens for concurrent use with molecular data. It makes it faster to reinforce the accuracy of the identification of a specimen, which is of vital importance from a biosecurity point of view. Also, having a good database or library of specimens and DNA sequences will help future identification of species be quick and accurate.

Is this Nemo?, it doesn't look like him but DNA
Barcoding could help determine whether or not it is
Photo by Harsha K R
There are some limitations to this method though. Not every fish that gets to the border will have come to NZ before. New fish will not have a DNA sequence in the molecular library or a voucher in the specimen library. Rupert and collegues identified this problem and suggested that using universal DNA sequence databases to figure out the identity of these novel fishes would overcome this. These databases are called Blast and Bold. In principle, the idea is good: you sequence the DNA of a fish of interest and test it against a global database of DNA sequences. If an incoming specimen fish wasn’t NZ's compiled specimen and DNA library, but had been sequenced somewhere and had been uploaded to one of the global databases, you could still identify the fish. However, the main issue with these databases is you often cannot confirm the accuracy of the morphological identifications, so a sequence that is identified as one species of fish, may be something completely different! It was found that 24% of the fish barcoded were not recorded on the most up to date aquarium trade reference list, with a quarter of fish sampled potentially misidentified. Therefore Blast and Bold databases should only be used when, a fish comes in that does not match anything in the library of currently known fish, which was the case.

As a practicing molecular ecologist I have a strong appreciation of the value of good genetic data, when used appropriately. I should be clear that, it is vitality important that we have an accurate and efficient tool for identifying fish so we can manage incoming fish specimens that may present a risk to our waterways and biota. DNA barcoding can be complimented with multiple methods to ensure we have the best toolbox for managing potential biosecurity threats and giving our fish front-line protection. Because you never know when you may end up finding Nemo!

If you want to find out more about DNA barcoding for biosecurity of ornamental fish check out the paper at this link http://www.plosone.org/article/info%3Adoi%2F10.1371%2Fjournal.pone.0028381

Source papers:

Collins RA, Armstrong KF, Meier R, Yi Y, Brown SDJ, Cruickshank RH, Keeling S, Johnston C 2012. Barcoding and Border Biosecurity: Identifying Cyprinid Fishes in the Aquarium Trade. PLoS One 7(1).

Collins RA, Armstrong KF, Holyoake AJ, Keeling S 2013. Something in the water: biosecurity monitoring of ornamental fish imports using environmental DNA. Biological Invasions 15(6): 1209-1215.

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