The boxfish.

Most of the time, I use this blog to blather on and on ceaselessly about all the things about life on this planet I find inescapably fascinating. While all of my exposition on killer fungi, badass birds, weird plants, or whatever obscure, bizarre, horrific, extinct monstrosity wandered into my search history that week is charming (obviously) and fun and all, I don’t often indulge in not only talking about the things that I think need to be shared, but things that are also very directly related to my scientific, academic interests. But, today I shall pander to myself and the relatively narrow realm that constitutes my research interests in the hope that you, dear reader, can push through the voluminous, insatiable outwards expansion of my own ego and acknowledge that my currently proposed study organism for my PhD research, the proud, doughty boxfish…is pretty goshdarned fucking cool.

While I plan on investigating certain nuances about the genetics and evolution of this special group of fishes, the topic of this post isn’t on the subtleties of things like gene flow between populations and speciation, but instead on an incredible, noxious, chemical adaptation that is unique to the boxfish.

But first…what exactly is a boxfish? Boxfish are small fish (between about 5 and 18 inches long, but most are at the low end of that range) that frequent the shallow areas of the warmer parts of the world’s oceans, like coral reefs and seagrass beds. They spend their lives passively pruning algae and small invertebrates like crustaceans, worms, and sponges off rocks and coral with their tiny, delicate mouths. They, as a group, are united in having a body made conspicuously rigid with hexagonal, bony plates fused together to form a hard, yet light-weight shell that encircles their interior, “real” skeletal framework. This shell (which has recently been used as bionic inspiration for automobile design) often has modestly rounded corners, and makes the animal distinctly rectangular in overall shape…hence the “boxfish” name (many species are also referred to as “trunkfish,” and there a some species with preposterously unintimidating horns called “cowfish“). This is an animal that is too hip not to be square.

So, this full-body shell results in the boxfish having a skeleton that essentially looks like a decapitated skull. Similarly to a skull, there are precious few holes in the cage of bone, and the formidable armor only opens up for the eyes, puckered mouth, fins, and tail to peek out into the water. When desiccated corpses of boxfish wash up on beaches, their remains resemble the forgotten, bleached craniums of ill-fated livestock out of a stereotypical, “harsh” cartoon desert.

Being a living tank confers some benefits. The most obvious of these is that it makes you really damn difficult to eat. Not too many predators jump at the opportunity to slam their pearly whites down on what is effectively the peach pit of the sea. Even for big, powerful, voracious, predatory fish, nomming on boxfishes is less like satisfyingly splintering a hard taco and more like trying to chew a kneecap; it’s a poor decision for all parties involved. Because of this heavy armor, boxfish aren’t exactly the fleetest fish under the waves. Boxfish are only able to propel themselves by using rapid undulations of their itty-bitty fins and exposed tip of their tail, as their entire body has been made inflexible by their bony shell. This is in contrast to most fish, which use strong muscles running the length of their bodies to move their tails side to side, torpedoing their streamlined forms forcefully through the water…and rapidly out of the toothy grasp of whatever is trying to eat them. Boxfish have made an evolutionary tradeoff, sacrificing speed and agility for enhanced defensive capabilities. Also, what they lack in power and evasive skill, they make up for with precision, being able to turn on a dime and dart into secluded overhangs and holes in the reef when pursued as a meal. As someone who has personally sought capture of these little creatures on many occasions, I can attest to their impressive maneuverability, leading to much frustration on my end. Frankly, it’s remarkable how quickly one can go from “aw, look at this cute little guy” to “oh, don’t you run away from me, you miserable son of a bitch” when you need to bag these buggers.

The strategy of hedging your reproductive future on simply being exceedingly unappetizing at the expense of athleticism is not an evolutionary route commonly taken by fish…but boxfish and all their close relatives have turned this approach into an art form. By “close relatives” I mean the order of fish to which boxfish belong, Tetraodontiformes. Tetraodontiform fish, almost without fail, have followed this unconventional path of taking the slow lane while arming themselves with a whole host of tools crafted by evolution to convince everything else in the ocean that, just like a questionably odoriferous bowl of tepid potato salad, they are a meal that just isn’t worth the pain. This coalition of fish, of which boxfish represent a single family (Ostraciidae), are “highly-derived”, meaning, in this case, that they exhibit a high degree of evolutionary modifications that are unique to that group; simply put, tetraodontiform fish are exceptionally, fundamentally different among their finned, gilled brethren. The group, over the course of its tens of millions of years of evolutionary history, has set along a curious trend of relentless reduction of all their fishy features. They’ve lost dorsal fins, many entire bones and portions of their skeleton (just little things…you know, like ribs), simplified their gill coverings, and shrunk their mouths (but kept their jaws big and powerful). They’ve condensed a mouthful of chompers into a handful of hard, strong teeth forming a beak-like shearing apparatus. They’ve even downsized the scope of their genomes (the entirety of the genetic material of an organism), multiple, independent times across several families, and one species, a freshwater pufferfish from Japan, has the smallest vertebrate genome currently known.

This overarching trend of maximum efficiency has been accompanied by not only the development of a wide array of body shapes, perhaps more diverse than any other order of fish, but also an impressively varied range of anti-getting-incorporated-into-the-food-chain measures. Examples abound, and tend to be grouped by family. Boxfish have teeth-unfriendly armor. Porcupinefish can bloat up to two times their original width, often lodging their spiny, spherical selves in the soft craw of whatever tried to choke them down. Triggerfish and their close relatives, filefish, can erect and lock into place sharp dorsal spines that make them very hard to remove from tight spaces within the nooks and crannies of the coral reef. One small family, the molas or ocean sunfishes, are covered in rough, sandpaper-like skin, and grow to such outlandish sizes that few things could tangle with them even if they tried. Pufferfish obviously have the ability to turn themselves into unchewable living balloons, but many species have tissues laced with a toxin so monstrously potent that even the most minuscule amount of exposure is more than enough to leave a human morgue-bound.

Boxfish hail from a line of fishes that are leisurely-swimming, anti-predator toolboxes. Tetraodontiformes, as an order, is a veritable Swiss Army knife of inventive evolutionary gadgetry; a fun-bag of bony plates, spines, levers, inflatable bodies, thick skin, and deadly chemical cocktails.

Adding to the extensive list above is one final trick up the tetraodontiform’s wet, salty sleeve…and it belongs to the fish with angles that’s hard to mangle…the boxfish. Like puffers and some species of porcupinefish, the boxfish is also armed with chemical defenses. It is perhaps no surprise then, that many species of both pufferfish and boxfish sport bright coloration as a passive aggressive warning to would-be diners shopping around the reef for the meal (an evolutionary phenomenon known as “aposematism”, which I outlined in other species very recently). While many species of boxfish are also known for potent differences in the coloration of males and females, both sexes show off conspicuous markings to advertise their toxicity, often consisting of brilliant stripes, reticulations, and spots.

So…big deal, right? Lots of things in the ocean are toxic or venomous and have warning coloration; everything from lionfish to sea slugs. What makes boxfish toxicity any different than, say, the toxicity of their close relatives, the pufferfish? The answer comes down to two things; 1) the toxins are completely different and are chemically very unlike almost all other known fish toxins, and 2) the toxins are used in an entirely different way.

Puffers, and most other creatures in the ocean that are endogenously toxic, defend themselves by embedding the compounds in their tissues, right in their very bodies. They saturate themselves with the poison, perhaps taking on a foul taste and/or sickening whatever animals are foolhardy enough to take a bite. But boxfish, when alarmed or actively being eaten, secrete their own toxin in a slimy mucus that oozes from specialized skin cells all over the fish’s body. The clear, toxic snot immediately disperses into the water column, creating an invisible cloud of death surrounding and trailing a very spooked little boxfish.

The force field of chemical ruination unleashed by the boxfish is well-recognized by tropical aquarium hobbyists, many of whom have learned the hard way about the potential consequences of raising these animals alongside other unsuspecting fish. The moment one of Boxie’s tankmates gets little too touchy-feely, the “oh shit danger danger danger!” trigger gets pressed and all hell breaks lose. The boxfish promptly sploogies out a coating of poison goo, and the stuff silently billows out, spreading throughout the tank insidiously and, initially, imperceptibly…..like a fart in an elevator. It isn’t long until the effects of the toxin start choking out just about every susceptible living critter in the vicinity. Under normal conditions, the toxin is released in the vastness of the ocean, with all its currents and fluid mixing, where the toxin eventually is diluted to the point of being functionally benign. But in the tiny, closed system of an aquarium, the lethal excretion becomes trapped in a small volume, creating an aquatic Dutch oven effect. There is no escape from the concentrated, toxin-infused waters, relative exposure continues to climb, and soon everything in the tank that is vulnerable goes belly up, like roaches trapped in a tented, bug bombed house. I’ve personally heard stories from aquarium owners who have come home to a watery, glass graveyard of floating Nemos…and a single boxfish quivering under a ledge of fake coral, hangdog expression betraying its role in that afternoon’s pescicide.

So what is it, exactly, about those nasty, mysterious, skin residues that would allow such a creature to evade predation in the wild…as well as inadvertently nuke an entire tank in captivity, wiping it clean of all complex life? How does this chemical weapon actually do its job?

The secret of how pahutoxin (what we’ve so far identified as the chief toxin in the mucus secretions, also known as “ostracitoxin”) actually moves itself from the surface of the skin, into the water column, and into places on other animals where it manages to screw things up royally, lies in its basic chemistry. Pahutoxin is a type of chemical compound known as a “surfactant.” Without getting into too much detail about how surfactants work at the chemical level, the general gist is that these compounds are often quite good at 1) dispersing in water and 2) making certain things that don’t dissolve in water suddenly very good at dissolving in water. A good example of a surfactant that you and I use (hopefully) every day is soap, or pretty much any other detergent, for that matter. Soap works by way of a number of chemical properties that allow it to make globules (called “micelles”) around the oils in grime and dirt, which normally repel water molecules and do not mix with good ol’ H2O worth a damn. The detergent effect of soaps comes down to the capacity to “cage” microscopic bits of insoluble grossness in tiny bubble-like formations, allowing it to be washed off whatever surface you want with the addition of water.

And just like how running water easily washes away the soap-and-filth slurry from formerly dirty hands, the ocean’s water readily spreads pahutoxin out and away from whatever boxfish is currently shitting its tiny, vibrantly-colored pants, and right into the mug of the hungry, predatory, and now very unlucky pursuer. It’s also worth noting that nothing about the general appearance of the pahutoxin-saturated mucus conceals the fact that it behaves similarly to detergents. I’ve seen on numerous occasions extraordinarily unhappy boxfish pulled out of the water, decorated so heavily with sudsy mucus that they look like they were just scrubbed down with fucking Dawn. It also comes as no surprise to me that while pahutoxin is certainly a unique chemical among fish (as far as we know), it’s thought to behave similarly to a group of toxins described in sea cucumbers, which are, chemically, “saponins”…which are distinctly known for tending to create lots of bubbles and suds.

So, surfactants are specifically good at breaking up things made of oils, fats, and waxes…things that aren’t normally soluble in water. The membranes that make up the barriers that keep the innards of animal cells nice and contained? They’re, conveniently, made out of a bilayer of lipids (a group of molecules that contain fats, waxes, a lot of fat-soluble vitamins, etc.), and are therefore theoretically susceptible to a surfactant’s detergent properties. It was thought for a long time that detergent properties of pahutoxin on the cell membranes themselves was the primary way in which the toxin hurt aspiring attackers. But studies in the last decade or so have hinted at a far more nuanced and complex mechanism of action in pahutoxin, where there may be very specific sites on the gill membrane cells of fish where this toxin binds….sites that are not shared by relatively pahutoxin-resistant boxfish, explaining, in part, the reported, temporary resistance to the toxin’s deleterious effects among boxfish.

And what, exactly, are these effects? They are…well…pernicious, to say the least.
Pahutoxin has an effect that what we would call “hemolytic”, meaning that the toxin tends to pop red blood cells like goddamn water balloons (“hemo” referring to blood, “lysis” referring to rupturing). Given that there seems to be particularly potent effect on the gill tissue of “enemy” fish, a region where there’s a lot of blood vessels and very important exchange of oxygen and carbon dioxide going down, all of which is necessary for a fish to, you know, not immediately die…this makes a lot of sense as a good way to keep big, ravenous fish from doing what they love to do…eat small fish like boxfish. Although pahutoxin appears to have evolved to specifically combat gilled predators like sharks or groupers, adverse effects from the toxin have been reported in mammals in laboratory experiments, and there are reports of severe poisoning in humans who attempted to eat cooked boxfish. But, the main mechanism appears to be one honed by evolution to disperse widely and make its way into the vulnerable gills of an unfortunate meal-seeker, where it promptly takes to exploding life-giving red blood cells left and right, incrementally cutting off oxygen from reaching the rest of the fish’s body. If the attacker can promptly leave this smog of death, it suffers lasting, irreversible effects, but can potentially live. If the predator tries to eat the boxfish, or is trapped in a tank with one releasing its toxins, the pahutoxin’s effects overwhelm the ability to take in oxygen, and before long, the unfortunate creature meets its end at the hands of agonizing asphyxiation…all at a concentration in the water as low as only 10 parts per million. For some visualization on just how impressively potent this is, consider that a concentration of 10 ppm can be reached by squeezing several drops from an eyedropper into a keg of beer. Spitting out a modest swish of mouthwash into a 660,000 gallon, Olympic-size swimming pool also achieves a 10 ppm concentration.

It’s also possible that boxfish don’t even make the toxin themselves. It’s may be that they “contract” it out to special bacteria that they harbor in the skin cells that make the poisonous mucus. It wouldn’t be all that unusual, considering that they are so closely related to pufferfish, which have shown on numerous occasions, and in multiple types of tissues, that they contain bacteria (most notably Vibrio) which just so happen to manufacture their famously deadly tetrodotoxin (TTX), and are likely the primary source of this toxin in these fish; an amazing symbiosis of “house me in your body, and I’ll help obliterate anything that tries to eat you…I mean “us”, right buddy?”. A species of Vibrio has been found in the mucus-producing cells of at least one species of boxfish, but at this point, it’s not clear whether this toxin, very different from the more famous, and dangerous, TTX, is actually being produced by the bacteria.

Either way, boxfish are remarkable fish, and have…cornered…the market on a particular method of chemically defending themselves. This method, which has more in common with the acrid sprays of landlubbing skunks than with anything fish in our oceans have evolved, is truly unique.

It’s important to note that while we may not yet completely understand how pahutoxin works, we should acknowledge that pollution of our oceans with compounds that chemically mimic the ways in which pahutoxin interacts with cells and specific binding sites…specifically detergent pollutants like soaps, which can accumulate in coral reef habitats from nearby human habitation and subsequent waste runoff…has the potential to disrupt the way these chemicals normally interact. Detergent pollutants in the environment may block receptor sites for pahutoxin…or yet undiscovered surfactant chemicals in our oceans…and restrict the effectiveness of the toxin in a natural, anti-predator setting. If pahutoxin can’t do its job, then that puts boxfish, or other organisms that depend on natively-produced surfactants working like they had evolved to, at a greater risk of increased exploitation by predators, and eventual population decline.

Image credits: introduction image, very colorful boxfish with yellow spots (Ostracion meleagris), hiding boxfish (credit Christie Wilcox), yellow boxfish (Ostracion cubicus)

© Jacob Buehler and “Shit You Didn’t Know About Biology”, 2012-2014. Unauthorized use and/or duplication of this material without express and written permission from this blog’s author and/or owner is strictly prohibited. Excerpts and links may be used, provided that full and clear credit is given to Jacob Buehler and “Shit You Didn’t Know About Biology” with appropriate and specific direction to the original content.