I had a lot of great experiences during my first year of university, even in my introductory biology classes.
For example, I can remember listening to Dr. Steven Newmaster, a taxonomist who was hooked on plants, recount a story about going to a hardware store to buy wood for a remodeling project for his house. The salesperson who was helping him assured Dr. Newmaster that all the wood this well-known hardware store chain was selling was ethical, meaning that it was devoid of any endangered species of trees.
Doubtful, he took a sample of the wood he wanted to purchase back to his lab to analyze it. Sure enough, it came from an endangered species.
Dr. Newmaster deplored living in a world where it was impossible to easily verify the claims made by supposedly ethical companies like this hardware store. He told us of how he dreamed of a day when anyone would be able to bring a portable DNA barcode scanner into a store, allowing them to identify any species, plant or animal, within minutes.
After class, I continued to think of the many applications such a device might have. One could go outdoors and scan any plant to find out what it was. The inhabitants of any anthill would be instantly identifiable. One could scan the contents of a tuna can to make sure that it was devoid of dolphin meat or one could use this machine to scan a 100% beef hotdog to see if the label was accurate. A customer could go into a sushi restaurant and make sure that the expensive red snapper on the menu had not been substituted by a cheaper kind of fish.
All these exciting examples kept popping up in my head. I still get shivers when I think about it. The building of a DNA barcode database has barely begun, but perhaps I will get to see the results of this endeavor in my lifetime.Regardless, because of this experience in that biology class two years ago, I got very excited when I saw the following Le Fresne et al. (2011) journal article entitled “Application of Denaturing High-Performance Liquid Chromatography (DHPLC) for the Identification of Fish: A New Way To Determine the Composition of Processed Food Containing Multiple Species.”
In 2004, a study by Marko et al. determined that nearly ¾ of the red snapper (Lutjanus campechanus) sold in the U.S. belongs to another species. Mislabelling fish products, whether accidentally or intentionally, can have major health, economic and ecological consequences.
This is a big problem for scientists who monitor endangered species by estimating fish stocks. Individuals with allergies wishing to avoid certain species are also at risk because of faulty labeling.To remedy this situation, Sophie Le Fresne and her team of researchers set out to come up with a fast and efficient method of identifying the species contained in heat-processed food products, mainly surimi-based products.
Surimi is a term used to designate a thick fish paste used mostly in asian foods, such as sushi. Its manufacturers make it by rincing and dewatering minced boneless fish meat in order to concentrate the myofibrillar proteins of the meat. After this process, the resulting paste can be frozen or used to make products such as imitation crab sticks, an essential element of the california roll.
To make crab sticks, the manufacturers add a layer of red dye, some enzymes, albumen (egg white) and gluten. Once in crab stick form, the fish product is unrecognizable, making the external identification of the species it contains virtually impossible.
There are many existing methods, mostly protein based, to identify fish species. These methods include iso-electric focusing and reverse phase high-performance liquid chromatography (HPLC). These techniques cannot be used for the heat-processed crab sticks, however, because they only work when used on samples that have undergone processes that do not affect the integrity of the fish proteins.
Scientists have developed other techniques, such as DNA fingerprinting techniques. DNA is not damaged by heating or extended periods of freezing, but this technique is hard to use with impure samples, that is, when a sample contains more than one species of fish. Numerous techniques address these problems but each of them pose their own unique difficulties, such as being limited for use on a single family of fish.
The Method: Denaturing HPLC
In order to avoid all these difficulties, the researchers propose using Denaturing HPLC (DHPLC), a process that allows each species to have a characteristic DHPLC profile. This technique can be used to generate complex profiles that can include multiple, unrelated fish species.
The first thing the scientists had to do was create a DHPLC profile library made up of the fish species commonly used in surimi. The researchers selected 13 species, including atlantic salmon, flying fish and jack mackerel.
They obtained the DNA sequences of the cytochrome b gene of each species courtesy of the National Center for Biotechnical Information and Fishtrace. They then extracted DNA from pure fish samples and used universal primers to generate amplicons, amplified portions of the cytochrome b gene (cyt b gene). The cyt b gene is highly conserved and varies from species to species, making it ideal for species identification.
A sample containing one amplicon from a single sequence generates one characteristic peak that scientists can use to identify species of fish. In a sample containing many different types of fish, multiple peaks that can be used estimate the number of fish species it contains.
The researchers subjected the resulting amplicons to DHPLC analysis. They compared these referent DHPLC profiles to the DHPLC profiles of ten samples of different commercially available crab sticks.
By comparing the peak patterns and the fraction collection, the researchers were able to estimate the number of fish species contained in the commercial crab sticks.
To identify the specific species contained in the crab sticks, the researchers used “similarity analysis”. If the crab stick profile was similar in aspect and retention time to one or more referent profiles, then they could infer the identity of the fish in a sample.
Here is an example of a crab stick DHPLC profile:
In this profile (black line), there are three major peaks, indicating that at least three species of fish are present within the crab stick. Two of the peaks match those of Theragra chalcogramma (Alaska pollack) and Pennahia sp. (croaker). Unfortunately, the third peak matches the peaks of four referent fish species so the researchers were unable to identify it using this method. Further sequencing revealed that the third fish species was jack mackerel (Trachurus murphyi).
All the crab stick profiles contained between three and seven major peaks, including those that the manufacturers claimed contained a single species. The researchers were unable to identify certain peaks because the referent library was too small.
In order for this method to work, the library must contain the profiles of all the possible fish species that manufacturers use to make commercial crab sticks, a large undertaking, particularly given that the depletion of certain fish stocks has caused surimi manufacturers to resort to using new species of fish to make the paste.
As you can see, this method has its limits. More often than not, the peaks generated matched more than one of the referent peaks.
Once perfected, however, DHPLC could be used to identify species in other heat-processed fish products such as “fish fingers”.
The researchers note that all but three of the ten crab stick manufacturers refrained from stating the species of fish contained in their products, preferring the use to generic term “fish meat” instead. We are still far away from the day when consumers will be able to use this method on their own, but its use in enforcing regulations involving the use of endangered species seems attainable, which is not quite as shiver-inducing as a portable DNA barcode scanner, but exciting nonetheless.
Le Fresne, S., Popova, M., Le Vacon, F., & Carton, T. (2011). Application of Denaturing High-Performance Liquid Chromatography (DHPLC) for the Identification of Fish: A New Way To Determine the Composition of Processed Food Containing Multiple Species Journal of Agricultural and Food Chemistry, 59 (23), 12302-12308 DOI: 10.1021/jf2030242
Marko, P. B.; Lee, S. C.; Rice, A. M.; Gramling, J. M.; Fitzhenry, T. M.; McAlister, J. S.; Harper, G. R.; Moran, A. L. Fisheries: mislabelling of a depleted reef ﬁsh. Nature 2004, 430, 309–310.doi:10.1038/430309b