What’s In a Name: Poison Monks and Fugu Evolution
One of Japan’s most notorious delicacies, Fugu (a.k.a. Pufferfish), made recent news when two people fell ill after accidentally consuming Fugu that had been mislabeled as Monkfish. Perhaps the Hong Chang Corporation who mislabeled the fish was confused by the fact that both Anglerfish (Lophius piscatorius) and Angel shark (Squatina squatina) are referred to as Monkfish. The bad fish snuck its way into soup imported by Hong Chang, and is thought to have been in mislabeled boxes shipped to California, Illinois and Hawaii. The FDA is advising caution with fish sold as Monkfish.
The Japanese have been eating Fugu for hundreds of years and its bones have been found with human remains dating as far back as 2300 years. But even though its deadly reputation has made Fugu popular with the common man, Fugu is the only food forbidden to the Emperor for his own safety. Although newer to the western table, Fugu’s reputation has preceded it. In an episode of the Simpsons (“One Fish Two Fish Blow Fish Blue Fish”) the main character Homer is given 24 hours to live after eating Fugu.
As the mislabeling victims learned, Fugu (short for Takifugu rubripes) is not your mom’s Monkfish, though its notoriety is hardly new. Most notably some of the tissues of Fugu harbor a poison called tetrodotoxin, a very potent neurotoxin with no known antidote. Tetrodotoxin is an alkaloid whose shape and chemical properties mimic that of a hydrated positively charged sodium ion (see Figure 1).
This becomes important in the context of conducting the nerve impulses that regulate everything from lifting a finger to pumping your blood. Nerve impulses are propagated along nerve cells like electricity along a wire. When there is no impulse being sent, positive and negative ions are distributed between the inside and outside of the nerve cell such that there is a net negative charge (approximately -70 mV) inside the cell and positive charge outside. When an impulse occurs, channels made of proteins embedded in the cell membrane open like doorways, allowing the sodium ions to rush into the cell. This redistribution of ions, called depolarization, discharges the “resting potential” (-70mV) of the cell past 0mV to +40mV and an impulse is propagated to adjacent resting sections along the nerve cell membrane. After being open a short time, the channel transiently enters an “inactivated state”.
Tetrodotoxin prevents depolarization by binding part of the channel; a ring shaped part of the toxin then blocks the channel opening, interfering with the rush of sodium into the cell. If we look at the protein’s binding affinity for sodium versus the toxin we find that while sodium binds the channel reversibly for nanoseconds at a time – a quick kiss on it’s way through the pore, the toxin binds and holds for tens of seconds, essentially inactivating the channel. That could be a problem if you’re an unfortunate diner trying to send nerve impulses, say to your diaphragm. Small doses of it give the mild tingle on the tongue sought by voyeuristic food snobs, large doses land you at the morgue.
A fortuitous mutation in the gene encoding the channel proteins of T. rubripes prevents tetrodotoxin binding, and allows the fish to house this deadly substance in its tissues unaffected. It is now accepted that the toxin is actually produced by bacteria ingested by the fish (Matsumura 1998).
But that isn’t the only important difference between Monkfish and Pufferfish. The Monkfish’s genome, estimated to be over 1000Megabases (Mb) based on DNA content of its cells (Hinegardner 1972), is much larger than that of Takifugu rubripes (400 Mb). In fact, T. rubripes has the smallest vertebrate genome (Venkatesh et al. 2000).
Organism Genome size (Mb) Human (Homo sapiens) 3000 Mouse (Mus musculus) 3000 Chicken (Gallus gallus) 1200 African Clawed Frog (Xenopus laevis) 3100 Zebrafish (Danio rerio) 1700 Takifugu rubripes 400
Table 1: Genome sizes of model organisms. From: Venkatesh et al. 2000.
This unusually small and compact genome has made T. rubripes a useful genome to sequence as a reference for other vertebrate genomes. Other genomes, notably the human genome, contain a lot of non-coding DNA, DNA that doesn’t code for a gene. The human genome is only 3% coding sequence, whereas T. rubripes is 17% coding sequence. This compactness not only makes sequencing easier, but also is important in the context of T. rubripes phylogenetic relationship to other vertebrates. The vertebrates listed in Table 1 descended from a common ancestor, a bony fish, about 450 million years ago. The bony fish, or Osteichthyes, gave rise to two types of fish, the lobe-finned (sarcopterygian) and the ray-finned (actinopterygian) fish. Mammals evolved from the lobe-finned fish and T. rubripes, and other teleost fish evolved from the ray-finned fish (see Figure 2). Because T. rubripes diverged from mammals so long ago, stretches of DNA sequence common to both are likely to be important as they have been retained despite millions of years of evolution. In particular there has been much research interest in non-coding sequences that been retained between mammals and teleost fish, suggesting that these sequences may be important for gene regulation (Venkatesh et al. 2000, Elgar 1996).
Perhaps the most obvious use of the Fugu genome has been to look at how genome sizes have increased and decreased as it evolved away from us and onto our plates (Froschauer 2006). An obvious question is whether species closely related to Fugu have similarly sized genomes and whether they inherited this small size or it was generated by a high rate of loss from an originally larger genome. Speciation, the process by which new species are born, is often accompanied by genome duplication events. When a genome duplication occurs the number of copies of the genome per cell doubles. This allows the cell to keep a “back-up” functioning copy of each gene while mutating the other copy making faster evolutionary change possible. There is a lot of interest in when genome duplications occur and how these relate to the birth of new species and new mutants in important genes. There is now convincing evidence that the ancestor to the teleost fishes was tetraploid (had four genome copies compared to the two copies in humans), suggesting T. rubripes has undergone significant reduction and streamlining in its DNA. Figure 2 shows the phylogenetic relationship of representative organisms. 1R, 2R and 3R indicate the estimated time of genome duplication events. During the divergence of mammals from teleost fish one such event is estimated, giving diploid mammals and a tetraploid ancestor for teleosts.
One thing is for certain: the western gourmands aren’t quite ready to eat Fugu at all costs. As of 2003, only 17 restaurants in the United States had chefs licensed to serve Fugu. This pales in comparison to Japan where Fugu can be had in most big cities. This doesn’t mean the Japanese don’t take their poison seriously, though. To serve Fugu a chef must pass an elaborate test consisting of a 2 to 3 year apprenticeship followed by a written test, a fish identification test, a preparation test and the final test: eating your own work. Perhaps it’s the Japanese fascination with the dramatic, illustrated in this haiku (I’m guess the syllable count works in Japanese) by Japanese poet Yosa Buson (1716-1783):
I cannot see her tonight.
I have to give her up
So I will eat fugu.
The Mad Gastronomist
It has been said that The Food Network is becoming the new MTV, and it is clear that there has been a surge in the popularity of all things food and cooking related. The casual weekend chef now has an almost unlimited amount of instructive information, and as their culinary acumen becomes more sophisticated, so too does their level of investigation. At this point, it is no longer satisfactory for cooks to ask “how do I bake a cake?”; the question has now become “how do these ingredients interact to result in a delicious cake?” To answer this question you have to turn away from the cookbook and bury your nose in a scientific text book. Or you could just read this column.