The Creative Works of Andrea Freeman
Stalking the Wild Tardigrade
by Andrea Freeman
I spent the last few weeks stalking bears in the wild. I combed the hills, traipsing through forested glens and moss-laden glades looking for their lairs. There was no need to observe the customary safety precautions recommended for outings in bear country— bells, whistles, food secured and hoisted up a tree, etc.— for the bears I was looking for are harmless. In fact, they’re microscopic. Known as ‘Water Bears’, they range in size from 0.005 mm to 1.7 mm in length, the largest being only about the size of the period at the end of this sentence. Those giants are the exception; most average between 100 to 500 microns, so are completely invisible to the naked eye. Minute they may be, but they’re memorable in their own right. Once seen under a microscope, they’re not easily forgotten. They amble along on four pairs of stubby legs, that support a chubby, cylindrical, 5-segmented body and a tapering, down-turned head complete with sensory eyespots1. Their lumbering gait and stocky build give them the appearance of tiny bears, which is how they earned their common name. Alternately, they’re called Moss Piglets, another descriptive appellation bestowed upon them by their admirers. More formally, they’re known as Tardigrades, and unique creatures that they are, have even been assigned their own phylum— Tardigrada, which is Latin for ‘slow stepper’. True to their name, they plod along at a modest pace; their maximum rate of progress has been clocked at 17.7 centimeters per hour under optimal conditions. Slow pace or not, they’ve made great strides in earning a notable reputation for themselves. For they are the “supermen” of the invertebrate world, exhibiting death-defying behavior that is rivaled by no other. But a little background information is in order first, before trumpeting their feats of greatness. The first tardigrade was described in 1767 by a pioneering microscopist named Eichhorn; prior to this, they were simply referred to as ‘animalcules’ along with the other invertebrate denizens of the micro world. Since their official discovery, more than 750 species have been described, some only recently. There are, no doubt, many others that have yet to be detected, particularly those that dwell on the deep sea floor, a region still virtually unexplored, but known to be inhabited by their kind. All tardigrades are somewhat aquatic, in the sense that they require water around their bodies in order for locomotion and gas exchange to occur. Yet a great many live in the terrestrial environment. A seeming contradiction, but they’ve found a way around it. Technically, the land-dwellers occupy what are referred to as limno-terrestrial habitats2. Their favorite haunts are the films of water on mosses, lichens and liverworts, which provide a succulent den of moist cushions in which to languish. However, not all live in such luxurious surroundings. They’re also found in more rigorous settings, such as in the sediment and algae in and around lakes and streams, as well as in the sandy bottom of marine environments, both deep and shallow. They can also turn up in roof gutters, leaf litter, and the cracks between paving stones. Some enterprising marine species are commensals on the gills of mussels or on the epidermis of sea cucumbers and barnacles. “To see a world in a grain of sand...” is a line from a William Blake poem. So one does when peering into the realm of the tardigrade. A clump of moss becomes a lush, tropical forest; tiny stems and leaves become towering trees with tapered spikes jutting out in dense, tangled profusion. Deep canyons and crevasses loom precipitously amidst the sediment that adheres to the roots. Minuscule mites, some with Maori-like designs adorning their carapaces, lurk between the fronds. Others look like semi-precious stones of smooth, opaque carnelian or jade embedded in a setting of filamentous filigree. Looking like candy caterpillars fashioned out of sugar-crystals, tiny springtails tiptoe delicately upon the water droplets, or without forewarning, spring spontaneously into the air to cover more ground. In the pools and rivulets of water in and around the islands of moss, tiny flask-shaped protozoa dart about, contracting and expanding with rhythmic pulses to propel themselves along. Others jettison around like animated apostrophes in a rush to find something to put into quotes. Nemertean worms slither by like water snakes. Rotifers spin along, their crown of cilia beating so fast that it gives the appearance of a whirring wheel atop their transparent bodies. This verdant moss jungle teems with life; a tranquil utopia it is not. Some of the aforementioned meiofauna are formidable foes for a tardigrade. In spite of being covered in a chitinous cuticle, as well as being equipped with claws, the water-bears, slow-steppers that they are, are often engulfed as prey by amoebas or nibbled up by the nematodes that share their Lilliputian world. Though combing through a magnified section of moss will sometimes reveal only a solitary individual, or perhaps none present at all, (which I disappointedly often found to be the case), occasionally a tardigrade metropolis will be encountered. They can occur in high densities of 300,000 per square meter in soil, and more than 2,000,000 per square meter in moss have even been tallied. But not by me. I seemed to encounter the lone individual more often than not. I began thinking of the loners I met as the ‘Turret Tardigrades’, akin to the artists and poets who sought refuge in themselves and their creative works, by living alone in their lofts or wandering the countryside as itinerant wayfarers. Though they’re not artisans in the literal sense of the word, preferring a simple life of feeding, molting and mating, tardigrade artistry is expressed quite elegantly through their body art. Particularly by the Heterotardigrades who have armoured plates covering their backs3, arranged in geometric configurations reminiscent of an African mask, each plate adorned with distinctive patterns and designs. The phylum Tardigrada is divided into two classes and the presence or absence of these plates is one of their main distinguishing features4. The two classes, the Heterotardigrada and the Eutardigrada, are referred to colloquially as the ‘armored’ and ‘naked’ forms, respectively. There are members of each found in both marine and limno-terrestrial habitats. Though both are ‘slow-walkers’, the unencumbered Eutardigrades mosey along at a faster clip than those lugging around suits of armour. That’s the price they pay for fashion. The elaborate design-work and mix of textures on the plates worn by the Heterotardigrades look like they were taken straight out of a Gustav Klimpt painting. The array of textures and designs, some bumpy, some smooth, can include hexagonal honeycomb-shaped cells, a pattern of empty circles or circles with a bulls-eye in their centers, a background of small dots with pear-shaped islands interspersed, or an assemblage of random shapes that fit together like jig-saw pieces. Even in a given species, the texture of the plates can be highly variable. In addition to variations in their size and texture, the plates may be different sizes, fused or subdivided, further embellishing the complexity of the mask motif. Adding even more flourish to the look, are the assortment of spines and filaments that spring forth out of the animal’s back in varying size, number, and location depending on the species. Tardigrades can also be a colorful lot, some species being very brightly colored, others less so. Body coloration is largely determined by what they eat; for instance, the carotenoids in many lichens produce a beautiful bright orange in those who feast upon them. Built-in pigments in the cuticle, and granular bodies in the hemocoel also contribute to the body color. Non-marine tardigrades, particularly, can sport a wide spectrum of colors; some are shades of pink, purple, green, red, orange, yellow, gray, brown or black. Some are even striped. By contrast, I encountered one that was colorless and almost translucent. Being soft-bodied, very few Tardigrades have left a trace in the fossil record. To date, the only existing specimens that have been found are a single eutardigrade species (Beorn leggi) and a poorly preserved, unnamed juvenile tardigrade. Both were found in Cretaceous amber, so they at least date back that far. Based on the appearance of these ambered representatives, there seems to have been very little change in tardigrade morphology over the past 60 million years. They came up with a body-plan that worked and they stuck with it. Tardigrades have a remarkably sophisticated physiology for one so small. Despite the wide range of aquatic environments colonized by tardigrades, from hot springs to polar pools, from water-film on bryophytes to lakeside algae, from deep oceans to ephemeral ponds, the group displays surprisingly little variation of internal organ structure and organization4. They have a brain and a well-developed nervous system with both slow and fast nerve fibers. Sensory receptors in the form of eyespots and tactile cirri on their heads and legs aid them in navigating around the varied terrain of their microhabitat. And they’re muscular little fellows, too, having bands of both striated and smooth muscles6 . They don’t have a respiratory or circulatory system, but get along fine without them. Gas exchange occurs by diffusion across their body wall, which is why being enveloped in a film of water is so important to them. Their feeding apparatus is fairly complex, and rightfully so, since feeding is one of their specialties. In a number of species of Heterotardigrade, the mouth is positioned at the end of a retractable mouth cone, but for the rest, a stationary mouth works just fine. From a Tardigrade mouth, regardless of model, extends a pair of sharp oral stylets, that may be straight or curved. These are pushed out through the buccal cavity (mouth region) by a series of muscles, and then used as precision tools for piercing the cells of their food of choice. Stylet-straw inserted, they enjoy a smoothie at the makeshift juice bar they created, sipping up the fluids contained therein. Suction is provided by contraction and relaxation of the array of muscles that line the pharyngeal apparatus. Though many tardigrades are herbivorous, and suck the juices out of algae, moss, and lichen cells, some are more catholic in their appetites and use their stylets to puncture animal cells, as well. Soil-dwelling species may feed from a menu of bacteria, algae and decaying plant matter or may be carnivorous on rotifers and nematodes and sometimes even each other. Depending on the culinary fare to which they’re inclined, their head shape and mouth location will differ accordingly. Predatory and omnivorous tardigrades have a terminal mouth, whereas the mouths of the herbivorous and detritivorous ones are located beneath their heads. The type of ‘toes’ found at the tip of a water-bear’s eight legs also vary in accordance with its lifestyle. These may be equipped with claws, pads or discs, of which there might be up to a dozen of any one kind. All types are used for clinging to strands of vegetation or particles of sediment, but those that sport claws might also wield them as weapons for both capture and defense. Tardigrades even have a specialized claw gland, which secretes the same type of material as composes their cuticle; the gland’s secretions form deep infoldings from which new claws emerge. Eutardigrades often have two double claws on each leg, and their size, shape and symmetry may vary, as well as their point of attachment. Of the external structures visible in eutardigrades, the claws are the most important for identifying one taxa from another. Periodic molting occurs throughout the life of all tardigrades. And when they molt, they undergo a major overhaul. It begins with the expulsion of the stylets and buccal tube. The entire mouth opening closes up, and feeding come to a screeching halt until the molting process is complete, and a new buccal apparatus is regenerated. The salivary glands move forward as the old cuticular structures are expelled. From this new place of prominence, the salivary glands control the regeneration of the buccal tube, the stylets and their supports. The claws are reformed during each molt, as well. But that’s out of the league of the salivary glands, and is handled by the claw gland previously mentioned. A juvenile tardigrade lacks an anus. An awkward situation, particularly since they have to wait until their first molt to expel waste from their intestine. It’s a right of passage when they finally get one, which doesn’t occur until the second or third molt. It’s sort of like going through puberty, especially for the Eutardigrades, for, in their case, the paired sperm ducts of the male and the single oviduct of the female open into the rectum. It’s not quite so critical for the Heterotardigrades, for their sperm ducts and oviducts open into a separate, pre-anally (and sometimes post-anally) situated gonopore, which is alluringly surrounded by a rosette of cuticular folds. Sexes can be distinguished externally in some genera by the relative lengths of their cephalic (head) appendages. Male’s are longer than females. Body length, itself, can be a sex indicator for some species, with males again being the longer of the two. Males may also display an extra spur or hook on the inner claw of the first pair of legs. Species in one genus have a tell-tale lateral bulge on the fourth pair of legs, which is flattened in the male and rounded in the female. In many females, the ovary takes turns with the midgut in occupying the internal body cavity. When one is there, the other isn’t. That’s one way to avoid unwanted weight-gain during pregnancy! This system is, in fact, designed to allow the female water bears to maintain their small body size. It also means they have no choice but to completely stop eating during periods of reproduction. It has been noted that females exhibit a certain haste in getting the egg-laying process accomplished, attributing this to the fact that reproduction has to be completed during periods of favorable environmental conditions, which can often be of short duration. An alternate explanation might be that they’re just hungry! Feeding thankfully resumes when the period of reproduction is complete. Eggs can be laid freely in the substrate (soil, sand, moss, leaf-litter etc.) or enclosed in the cast-off molt shell (excuvium) of the female. In some cases, the excuvium full of eggs is towed around by the female, who safeguards them. This is the only form of parental care demonstrated by the Tardigrades. The quantity of eggs produced by a female can vary greatly, even within a single species. In one species observed, the eggs the different females laid in their molted shells ranged between 3 to 35 in number. Hatching is accomplished by the water bear cub piercing the egg with its stylets and legs. Depending on the temperature of its surroundings, it can take from five days to one month for the young tardigrade to reach maturity. Interestingly, eggs laid in the excuvium are smooth and nondescript, whereas the eggs deposited in the substrate are elaborate works of art, resembling expensive Christmas-tree ornaments. They may be decorated with cones, turrets, disks, or goblet shapes covering their surface. Some might look like a many-pointed star; others like the surface of a blade of a cactus in bloom. The function of egg-shell ornamentation is unknown, but given the amount of energy that is no doubt invested in producing their complexity, it’s assumed that it’s not just art for art’s sake. A few hypotheses have been suggested regarding the survival value that an ornamented shell may afford: Their uneven surface may help to secure them in place in the substrate, preventing wind, water, or any other disturbance from dislodging them as readily. Or, their sharp protuberances might serve as a deterrent to predation by nematodes, or the adhesion of fungal hyphae by various species of fungus that are known to parasitize the tardigrades themselves. Or, the possibility of dehydration might be slowed down by virtue of water being trapped in pockets between the projections. Nobody knows for sure. It’s also possible that eggs laid in the molt-cases have no need for ornamentation, since the excuvium would serve the same protective functions as those described above. In any case, variation in egg-shell morphology is a key tool in identifying the eggs of different eutardigrade taxa and they’re beautiful to look at, while doing so. How mate location occurs is also not well understood, though it is known that females are actively sought after by males. It’s thought that their cephalic (head) sensory structures may play a role in finding a suitable mate. Coming upon a female to his liking, a would-be suitor woos the female by stimulating her with his lateral cirri. (Oh, those lateral cirri!) She might be desired by many males, however, in which case, several males may cling to the front part of the female with their front legs, each vying for the chance to mate. Even when disturbed, they maintain their tenacious grip, unwilling torelinquish their hold until the act is complete. In limno-terrestrial eutardigrade species, fertilization usually occurs inside the body of the female, whereas the marine species have external seminal receptacles. The male’s aren’t particular. Male’s are also known to ejaculate sperm into the female’s excuvium as she molts, so as to fertilize the eggs she has laid inside of it. Or they may spread sperm upon eggs a female has deposited in the sediment. It’s not strictly boy meets girl in the world of Tardigrade sex, though. Some species are bisexual, some are unisexual, others are hermaphroditic. Hermaphrodism, which is usually considered to be a primitive condition in other animals, only occurs in the more advanced eutardigrades, and in a single marine heterotardigrade genus. The advantage of being herm-aphroditic is that every individual in the population is able to lay eggs . The tardigrades that are unisexual reproduce through parthenogenesis. These have the reproductive advantage of being able to colonize a habitat more rapidly, since only a single animal needs to be present to found a new population. No need to wait to find a mate. The straight males, when hunting for a mate, are able to distinguish between a heterosexual female and an undesirable parthenogenetic one, even though externally they may look very similar. When presented with the two, the males show no interest in the latter. They’re no fools. When times get tough, Tardigrades prove even tougher. Their extraordinary ability to survive environmental extremes, far beyond what most other living creatures could endure, has gained them a place of prominence in the biological Hall of Fame. When conditions become unfavorable, as in periods of drought or severe cold, they go into a state of suspended animation, known as cryptobiosis7. All metabolic activity stops— entirely— for as long as needed. While in this state, they become one of the most resilient animals known, impervious to almost any hardship. Conditions that would be instantly lethal to any active animal are no problem for the tardigrade. They’ve been known to withstand temperatures as high as 151 degrees Celsius, which is far above the boiling point of water, and as low as .008 degree Kelvin, which is on the brink of absolute zero. They can tolerate exposure to x-ray radiation that is more than 1000 times the amount that would be lethal for a human. They can survive exposure to a vacuum, and endure pressures of 6000 atmospheres, which is nearly six times the pressure at the bottom of the deepest ocean trench. At only half that pressure, the vast majority of even the most rugged bacteria are destroyed. Whatever the nature of the adversity, when it has passed, the tardigrades emerge from their great sleep, unscathed. Some tardigrades that were discovered in dried-up specimens of museum moss, were revived after being in their cryptobiotic trance for 120 years. Originally, the fact that there was no metabolic activity occurring, (which is usually synonymous with being dead), followed by the tardigrades’s seeming resurrection, led early scientists to refer to the phenomenon as anabiosis or “return to life.” They believed that the desiccated animal had actually died and when moistened, it came back to life. The name held, if not the belief, for 200 years. In 1959, David Keilin of the University of Cambridge finally gave a new name to the phenomenon, calling it ‘cryptobiosis’, which means “hidden life”. In other words, the tardigrades, though technically dead, are not technically dead. He felt it was a more accurate depiction of the phenomenon, since the suspension of metabolism was consistently reversible. They’d just adopted a ‘resuscitate me when it’s over’ approach to handling difficulties. Revival time is directly proportional to how long they’ve remained in the cryptobiotic state. Tardigrades that were cryptobiotic for just a few days have been observed to revive after only 10-15 minutes. A tardigrade that was found in a 20-year-old herbarium was given a drop of water and resumed a normal activity after three to four hours. The revival time probably varies from species to species, and is also affected by temperature, pH and dissolved oxygen. Akin to battening down the hatches, or donning foul-weather gear, the water-bears must make preliminary modifications before they can become invincible. They have a few different ways of achieving a cryptobiotic state, tailored to the nature of the problem that confronts them. When faced with the threat of dessication, as a result of their moss drying out or their ephemeral pool evaporating, they get a jump-start on the situation. They deliberately expel all the water in their tissues, invaginate their limbs, contract their body, and shrink into a shriveled barrel-shaped form known as a ‘tun’5. (A tun is the traditional name for a large cask of wine.) During this process, as their tissue dries out, their body’s water is replaced by a non-reducing disaccharide sugar called trehalose, which serves as a membrane protectant. The use of trehalose and another cryptoprotectant, called glycerol, help preserve the structural integrity of the cell organelles, which would otherwise buckle from the lack of water. These compounds are the ‘embalming fluids’ of choice, because they’re readily removed during rehydration. All this done, metabolism shuts down utterly. This type of cryptobiosis, initiated by desiccation, has its own special name— anhydrobiosis. If the impending problem is freezing temperatures, as faces the many limno-terrestrial tardigrades who live in polar regions, a similar sequence of tun formation occurs, at least superficially. In the case of cryobiosis, (the freezing-induced version of cryptobiosis), not all of the water is removed, since about 25% of the water in a cell is non-freezable. In anhydrobiosis, by contrast, even the non-freezable water is eliminated by desiccation. The biochemical adaptations necessary to tolerate freezing are also different. Ice growth needs to progress in a controlled fashion to prevent cellular damage from occurring. As such, it’s critical that numerous small ice crystals first be dispersed in the extracellular spaces. Certain proteins are released that seed ice-crystal formation, and anti-freeze proteins are also added to the extracellular fluids to ensure that the ice-crystals that form will remain at a small size, as long as necessary. Membrane protectants, such as trehalose, are also produced. Key enzymes, designed to respond specifically to low temperatures, get busy at increasing the synthesis of these cryoprotectants, while other enzymes are deactivated. Eventually, low-temperature-induced metabolic arrest occurs. The tardigrade is frozen stiff, but safe in its tun, it will be none the worse for the wear. When temperatures warm and the ice thaws, it will revive, stretch, and be as good as new. The tardigrades have still more environmental response tricks up their sleeves, because drying out and freezing aren’t the only hazards that befall them. Changes in salinity can also prompt them to retreat into a tun state. Though some species have a high tolerance to variations in salinity, such as those adapted to living in a coastal tidal zone, the bryophilous tardigrades prefer their salt levels to remain more constant. For them, it’s tun time when the salinity changes dramatically. And, of course, this has its own name, too. Osmobiosis is the term given to salinity-induced cryptobiosis. Another environmental endurance test that the water bears have to weather is that of insufficient oxygen. For instance, those living in dense carpets of the green alga Enteromorpha might find themselves in an oxygen-deprived state when the tide goes out and the algal thalli collapses. Under these circumstances, they employ a different strategy than the others. Here, they enter into anoxybiosis, a state in which their body swells, becomes turgid, and finally all movement ceases. Many freshwater (and some bryophylous) species initiate yet another survival tactic, but only when the environmental stress that confronts them is of a more gentle nature. Instead of a tun, they form a dark, thick-walled cyst. To achieve this, a tardigrade sheds its cuticle, but instead of discarding it, as it would in a normal molt, it recycles it to form the cyst wall. The contents of the midgut are defecated first to conserve on space. From here on, the tardigrade will rely on the food reserves it accumulated and stored in its body cavity cells prior to encystment. Its internal organs degenerate to some degree, but are reconstituted when circumstances are once again favorable. Since some metabolic activity can be detected during encystment, it doesn’t qualify as a form of cryptobiosis. Furthermore, cysts retain a substantial amount of water, so are much less resistant to wear and tear than tuns. Encystment is more like a form of quiescence, which is merely a slowing down of metabolism under unfavorable conditions, which many other animals also employ. The marine Arthrotardigrada, which are the most primitive order of tardigrades, don’t have the ability to enter into a cryptobiotic state. They are the only exceptions. But, in their case, it isn’t necessary. The marine environment is relatively stable, with no sudden fluctuations in temperature or humidity, so they have no need for radical retreats. Instead, they may undergo an annual alternation of morphologies called cyclomorphosis. They have a summer morph (short-sleeves and T-shirts) and a winter morph (long-underwear) that is resistant to freezing temperatures and low salinities. They remain active and motile in both forms, but only the summer morph is sexually mature. Over evolutionary time, some intrepid, pioneering tardigrades decided to venture onto land, going where no tardigrade had gone before. For the first time, they were faced with the possibility of drying out, a concept that had never even crossed their minds before. In order to occupy these new limno-terrestrial environments, they had to come up with a fool-proof plan. Cryptobiosis was the solution that evolution provided. It has an added advantage, too. Tardigrades that undergo cryptobiosis have a significantly longer lifespan than their marine compatriots. Normal life expectancy is only from eighteen to twenty months for those who have no down-time. This cryptobiotic survival strategy has worked so well, that tardigrades have been able to expand their range to the point where they now have cosmopolitan distribution. They’re found in microhabitats across the globe, on every continent, from temperate regions to tropical ones, and even survive in the frigid climes at the poles. They’re a common component of the microfauna of Antarctica. They’ve been found living on mountain summits as high as 6,600 meters, in lake bottoms as deep as 100 meters, and in the oceanic abyssal zone at a staggering 4,700 meters of depth. They seem to shy away from urban environments, however. City life is not for them. Urban pollutants, such as sulphur dioxide, may be the main deterrent, and responsible for their sparse distribution in such areas. Slow walkers that they are, how have they managed to cover so much ground? Being so small, they can be swept into the air by gusts of wind and transported from one body of water to another. Their ability to dehydrate allows them to be carried great distances like particles of dust. Or floating plants might ferry them across lakes and down streams, perhaps even carrying them to the sea, where ocean currents would carry them still further. Animals traipsing through a tardigrade habitat can inadvertantly serve as makeshift transport vehicles, as well. Soil or debris in which a colony of water bears resides might become attached to the fur, feathers, hooves or feet of any creature who happens by. Thus far, tardigrades have been found upon arthropods, mollusks, frogs and birds. Birds, especially, have likely been couriers in their long-distance dispersal. Tardigrades have been discovered on remote volcanic islands where only wind or birds could have carried them. Wet weather can also aid in their travels near and far. When raindrops splatter upon the ground, the tardigrades may be thrown into the air and trampolined to a neighboring spot. In Finland, some have even been found in the falling raindrops themselves. What a way to travel! It’s raining outside right now. Perhaps water bears are falling into my garden. I think I’ll go see what I can find.
© 2001 Andrea Freeman
Tardigrade SEMs (Scanning electron micrographs) courtesy of Science Photo Library.