The Puffer Fish Voucher Library
Keeping Food Resources Safe through DNA Science:
A Collaborative Inter-Agency Project
Story by Stephanie Guzik, (Volunteer Science Writer)
Puffer fish (Tetraodontidae), known most commonly around the world as fugu, are masters of self-defense. When threatened by a predator in the open ocean, they blow up like a balloon. Not only does this make them harder to swallow, but some species of puffer fish have sharp spines covering their body, and inflating themselves further exposes these spines. If the predator is not deterred and does take a bite, it may be in for another unpleasant experience. Some species of puffer fish have powerful neurotoxins (tetrodotoxins and saxitoxins) in their flesh and organs, which if ingested can incapacitate the predator. Together, these defense mechanisms provide a clear warning—don't eat me.
Despite the warning, some species of puffer fish are considered a culinary delicacy in Japan and other countries. Under carefully controlled circumstances, puffer fish can be safe to eat. But ingesting even a small amount of their neurotoxins can cause serious illness including weakness, vomiting, dizziness, paralysis, and in extreme cases respiratory failure and death.
Not all puffer fishes are created equal, according to Jeff Williams and Diane Pitassy of the Smithsonian National Museum of Natural History Division of Fishes at the Museum Support Center (MSC). There are currently over 185 known species of Tetraodontidae. Some species, like those found in the waters around Japan, have their toxins relegated to specific internal organs. This makes the flesh safe to consume if the fish is carefully prepared by a licensed fugu chef. Other species, like some found off the coast of China and off the east coast of Florida, contain toxins throughout their body that cannot be removed or deactivated during preparation—making any part of their body dangerous to consume. Still other puffer fish species, such as those located north of Florida on the east coast of the U.S. and those aquacultured in Japan, do not contain toxins and are safe to eat.
This variation in toxicity led the U.S. Food and Drug Administration (FDA) to initially ban the importation of puffer fish in 1980. In 1989, after four years of complex negotiations with the Japanese government, the FDA again allowed the importation of select puffer products, but only under a controlled set of conditions. The FDA only allows the importation of a single species of puffer fish (Takifugu rubripes, a.k.a. Tiger Puffer) from a designated source in Japan, where specially trained and licensed fugu chefs prepare the fish. The safely prepared product is then sent through a single import site in the United States. From there, it is distributed to a select group of restaurants that are authorized to sell fugu.
Despite this regulation, FDA has investigated sporadic cases of puffer fish (fugu) poisoning of U.S. consumers. Even though these events are rare, the consequences can be life threatening. Some of the illnesses were traced to domestically harvested product and some to product brought into the country illegally, outside the agreement between FDA and the Japanese government.
So how can research scientists like Jonathan Deeds and Sara Handy at the FDA determine the source of the puffer fish? Furthermore, how can they determine if the puffer was a domestic fish from the coast of Florida that should not have been eaten or a mislabeled illegal import that should be investigated and potentially prosecuted?
The answer to these questions is two-fold: collaboration and DNA.
Researchers at the Smithsonian's Division of Fishes and the Laboratories of Analytical Biology, the FDA, and the University of Guelph have worked together, each playing an important role in determining how to identify the species of puffer fish when all they had was a sample of processed fish.
The collaboration between the FDA and the Smithsonian on the puffer fish project began in 2007 in response to several illnesses resulting from the improper importation of toxic Chinese puffer fish that had been mislabeled as monkfish to circumvent the FDA's import restrictions.
Before the collaboration, FDA scientists used a technique called protein isoelectric focusing to identify the species from which the fish filets came. In this technique, protein banding patterns are identified from the fish sample and then compared to protein patterns isolated from known fish samples to try to make a match. However, this method doesn't always clearly identify the unknown fish and furthermore, if the fish sample has been cooked, the identification can be far less successful. The first samples collected as part of the 2007 puffer fish poisoning outbreaks involved home cooked puffer soup that would have been unidentifiable using the old protein based method. Deeds turned to Williams and the Division of Fishes at the MSC, as well as Japanese puffer expert Keiichi Matsuura of the National Museum of Nature and Science in Tokyo, Japan, to begin building a regulatory puffer fish DNA barcode library. The barcode library would be used to identify puffer fish poisoning outbreak samples as well as potentially mislabeled and smuggled puffer products imported outside of FDA's established guidelines. As a result of this collaboration, the Division of Fishes today contains a large collection of puffer fish voucher specimens stored at the MSC. This extensive library of reference specimens is essential to accurately identify and make comparisons among different species.
The collaboration also includes Smithsonian's Lee Weigt from the Laboratories of Analytical Biology, and scientists such as Robert Hanner at the University of Guelph's Biodiversity Institute of Ontario, in Canada, whose specialty is using DNA barcoding in forensic cases. DNA barcoding utilizes species-specific differences in the DNA base sequence of a small portion of the mitochondrial DNA called the COI gene, short for cytochrome c oxidase subunit 1. For most species studied to date, there tends to be very little difference among individuals of a single species in that portion of the sequence and sufficient differences between different species, usually making it possible to identify which species a tissue sample came from.
With the resulting Smithsonian voucher library linked to the corresponding sequences in the library of DNA Barcodes, and the techniques available to the FDA's research scientists, any specimen—whether raw or cooked—can be analyzed to determine the puffer fish species used to make the meal in question.
The puffer fish voucher library is only one example of the scope of this inter-agency collaboration. With the voucher library and DNA barcoding techniques in place, other food safety and food labeling issues can now be examined. Numerous other state, federal, and even international agencies responsible for regulating food have since joined in collaborating with the FDA and the Smithsonian to build a regulatory fish standards collection and combat the growing problem of species substitution. Additionally, the regulatory fish standards collection continues to grow through the work of Smithsonian researchers, with the FDA funding expedition efforts by Williams and other scientists to collect specimens.
According to Deeds, the collaboration between the FDA researchers and the Smithsonian is essential, combining the resources and expertise of all the collaborators to form a cohesive inter-agency unit and a database with endless possible uses.
Acknowledgements: Sebastian Cianci (FDA) contributed to the preparation of this highlight.
Further information on puffer fish from the FDA
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