Phylum Tardigrada

     The minute size of tardigrades has contributed to their status as a lesser known phylum. As science unravels their mysteries, we are discovering that tardigrades are fascinating animals and present a unique opportunity to advance the fields of developmental biology, evolution, and ecology. They are very successful animals and exceptionally abundant, even after 600 million years of evolution. Much of their success can be attributed to their ability to undergo encystment or cryptobiosis. However, this discussion will not focus on dormancy strategies; this topic will be presented as a poster. Additionally, Hypsibius dujardini will only be mentioned where appropriate due to the scant amount of information on this species. Instead, this paper will focus on the known features of various tardigrades.



     Tardigrades are molting animals and can be broken down into two main classes. They are barrel-shaped with four pairs of lobed appendages. They possess a molting cuticle that must be coated with a thin layer of water for respiration and to prevent desiccation. They have a unique feeding apparatus, and a varied excretory system. Most species of tardigrades have pigment-cup ocelli. They have a cosmopolitan distribution and can be found in marine, freshwater, and terrestrial environments. Most species eat plants, but some are carnivorous. Defense structures are speculatory, but probably present. Tardigrades can reproduce sexually or asexually. Gonochorism is only found in marine species, while terrestrial species possess separate sexes, hermaphroditism, and parthenogenesis. Eggs are externally fertilized and develop in the shed exoskeleton.



Classification



     Tardigrades were first described in 1773 by Johann Goeze who called them kleiner Wasserbär (little water-bear) due to their bear-like movements. In 1777 they were given the name Tardigrada, which means slow walker. It was not until 1962 when tardigrades were recognized as their own phylum (Ramazzotti 1962).

Tardigrades have a long evolutionary history. Fossil evidence from rocks found in Siberia of tardigrades or their early relatives place their evolution in the mid-Cambrian (Pechenik 2010). This suggests that tardigrades were present near the Cambrian explosion. They belong to the super-phylum Ecdysozoa. Recent genetic analyses with 18S rRNA places them as sister group to the arthropods and onychophorans, though complete relationships are not yet fully understood (Figure 1) (Campbell et al. 2011, Garey et al. 1996).

 

    There are two major classes of tardigrades- the Heterotardigrada and the Eutardigrada. The heterotardigrades are armored and considered the most ancestral class (Nelson 2002). Within the heterotardigrades, two orders are recognized. The Arthrotardigrada contain all marine species with the exception of one species. The other order of heterotardigrades includes the Echiniscoidae. These are mostly terrestrial species that possess an armored cuticle produced by thickened plates (Nelson 2002). The Eutardigrada are unarmored. Two orders are recognized in the eutardigrades- the Apochela, which are terrestrial species, and the Parachela, which are mostly live in terrestrial and freshwater habitats. Over 1,000 species of tardigrades have been described, but estimates suggest there could be as many as 10,000 (Gagyi-Palffy and Stoian 2011).



Anatomy and Physiology



     Basic structure. Tardigrades are small metazoans with barrel-shaped bodies divided into five segments and four pairs of unjointed, lobed appendages. The clawed species have four to eight claws on their feet, which are mainly used for attaching to substrate. Other species have legs that terminate in sucking discs to increase their grip strength as they walk along a surface. Most tardigrades are less than 0.5 millimeters long, with the smallest ones less than .01 millimeters and the longest up to 1.5 millimeters. They come in many colors including white, red, yellow, purple, black, and green. Tardigrades are eutelic, possessing the same number of cells from birth until death. In order to grow tardigrades increase their cell size. All tardigrades have striated muscles and a lobed dorsal brain that connects to a nerve cord with fused paired ganglia (Nelson 2002).



Cuticle.  Like their arthropod relatives, the inner layer of the tardigrade cuticle contains chitin. However, the external cuticle lacks calcium and is soft and permeable. It is built from a protein called an albuminoid, which differs from chitin (Gagyi-Palffy and Stoian 2011). Variations in a tardigrades cuticle range from smooth, to spiny, to armored plates (Biodiversity Institute 2008). The cuticle lines their foregut and hindgut and is shed along with the rest of their molt (Pechenik 2010). Molting occurs every five to ten days throughout the tardigrade’s life with the total number of molts ranging from four to twelve (Nelson 2002). Body length increases in juveniles until their final size is reached (Walz 1982). Their thin, uncalcified cuticle makes their body very susceptible to water loss. They do not possess any respiratory structures, so to prevent desiccation and allow gas exchange through passive diffusion they must have a layer of water surrounding their body.



Feeding and Excretion.  Tardigrades are suctoral feeders. They eat using their buccal apparatus, pair of stylets, and muscular pharynx to pierce into plant cells, algae, or small invertebrates (Figure 2). They suck out the internal fluids using their pharynx as a pump (Nelson 2002). The stylets are lost when the animal molts, but new ones are secreted via glands on each side of the mouth (Barnes, 1982).



     The digestive system of tardigrades consists of an oesophagus, an intestine, a rectum, and an anus with most of the digestion occurring in the intestine (Barnes, 1982). Excretory organs are only present in eutardigrades (Bertolani 1987). Differences in size and methods of excretion also exist. Weglarska (1990) found several genera of moss-dwelling tardigrades that have larger excretory systems than freshwater species. Weglarska (1990) also notes the different ways excretion can occur: through molting (removes excretory granules), through the salivary glands during molting, through the midgut wall, or through excretory glands. The presence of malpighian tubules in tardigrades remains unclear.



Eyes. The tardigrade eye is best described as ‘inverse pigment-cup ocelli’ (Marcus 1929). Eyes are found in the Eutardigrada and the Echiniscodea and probably do not exist in other Arthrotardigrada (Marcus 1929). Unlike their distant relatives the eyes of tardigrades are not similar to the compound eye in arthropods, which are more complexly organized. They are also unlike the visual organs of onychophorans, which possess an optical vessel with a lens-like structure (Greven 2007).

     A large diversity in complexity and sophistication is seen in the tardigrade eye. The general tardigrade eye is composed of a single pigment-cup, a single cell with microvilli, and one or more ciliary cells used for mechano- and chemoreception (Greven 2007). The ciliary cells are most likely not used for photoreception and the eye probably lacks a lens. The pigment granules are either black, brown or red depending on the species and the amount differs in individual eyes. Pigments may not occur in some taxa. The eyecups are closely associated with the outer dorso-lateral lobe of the brain, which suggests they might be directly evolved from it (Greven 2007). However, Kristensen (1982) suggests an epidermal origin of the eye due to presence of epithelial cells around the eye. The eyes are either positioned anterior or posterior depending on the size and length of the outer lobe of the brain. Little is known about the tardigrade eye and many more studies are needed to determine their structure and evolution (Greven 2007).



Life History and Ecology



Distribution and Habitat.  Tardigrades are found all over the world- from the tops of the Himalayas to the ocean’s abyss and from polar regions to the equator (Kinchin 1994). Some species have a cosmopolitan distribution while others have a limited distribution and are rare or endemic. The wide distribution seen in some species is probably due to their ability to undergo cryptobiosis (Nelson 2002). Eggs, cysts, and tuns can be distributed by wind, animals, or floating plants (Gagyi-Palffy and Stoian 2011). In some habitats, several million tardigrades per square meter are found (Pechenik 2010).
Tardigrades are either found in marine, freshwater, or terrestrial environments. The low number of species occurring in marine environments is indicative of their ancient origin and diversity (Nelson 2002). The sub-tidal zone harbors many different types of tardigrades. Some are benthic and live in the mud, while others are epibenthic. Freshwater tardigrades are found in all types of freshwater habitats from lakes to streams to ephemeral ponds (Bertolani 1982). Many freshwater species live in littoral zone, but some live at deep depths. They can be found on algal mats in Antarctic lakes, or in “cryoconite holes” in glaciers. These are holes that are formed when radiation from the sun is absorbed by the accumulation of dark dust on the glacier. Most tardigrade species are terrestrial and live in moist habitats including soil, leaf litter, mosses, and lichens (Kinchin 1994). Hypsibius is one of the most common genera found among the mosses (Glime 2007). High species diversity is not just found on mosses, but in leaf litter and soil where species are associated with a specific habitat preference (Nelson 2002).



Lifespan.  Tardigrades live from three to thirty months- cryptobiotic state not included (Nelson 2002). Their lifespan is greatly increased through encystment or anhydrobiosis. Species in the moss-residing Hypsibius can live from four to twelve years (Nelson 2002).



Diet.  Tardigrades are important consumers and in many instances they are the top carnivores for their size. They feed on bacteria, protozoa, insect larvae, algae, mosses, and lichens (Glime 2007, Nelson 2002). Larger species can pierce and pump the fluids out of nematodes and rotifers (Nelson 2002). Buccal tubes and claws have been found in the gut of Macrobiotis, suggesting some tardigrades species prey on smaller tardigrades (Glime 2007). Tardigrades are eaten by spiders, mites, springtails, and snails (Fox 1966, Nelson 2002).



Defense.  The scant amount of information available on tardigrade defense either suggests defense has not been well-studied or tardigrades do not possess any defense mechanisms. Nonetheless, examining their anatomy suggests they probably use several structures for defense. The armored tardigrades (Heterotardigrada) may use their armor for protection from predators. Perhaps, this armor evolved once the heterotardigrades (Echiniscoidae) made the transition from marine environments to terrestrial habitats to protect themselves from a new predator or to retain moisture (Figure 4). Clawed species, in addition to using them to attach to substrate, may use these to attack a predator or defend themselves from a threat. The most likely defense mechanism, at least in carnivorous species, is the use of their piercing stylet to kill prey and suck out their body fluids.



Movement and Dispersal.  Tardigrades possess both active movement and passive dispersal. When attached to a substrate for reproduction or feeding, they are slow-moving and clumsy. Marine tardigrades colonize new environments by the use of the ocean’s currents, either by attaching to another organism or passively floating. Terrestrial species, however, may disperse as cysts or eggs in the anhydrobiotic state (Biodiversity Institute 2008). Their small size allows them to be blown by the wind or carried by freshwater (Figure 5). Similar to their marine relatives, terrestrial tardigrades can be carried by larger animals or in the mud or sand that adheres to their body (Biodiversity Institute 2008).



Reproduction



     Tardigrades have colonized most marine and limno-terrestrial habitats. The widespread distribution and variety of habitats that tardigrades are found in is facilitated by the several modes of reproduction they possess.

Marine Tardigrades.  Marine species are always gonochoristic, which is considered the norm in tardigrades (Bertolani 2001). Separate sexes guarantees recombination, where as asexual reproduction does not. In marine tardigrades, males are usually smaller than the female and are most likely semelparous and breed at the end of maturation (Bertolani 2001). In contrast, females are iteroparous and produce eggs throughout their lifetime and in association with molting (Bertolani 2001). Eggs are externally fertilized in two “external cuticular seminal receptacles” that serve the function of storing sperm (Bertolani 2001). After sperm is deposited, the receptacles are molted with the cuticle and the eggs develop in the molt (Figure 6). The absence of parthenogenic and hermaphroditic species in marine environments suggests that the chance that males and females meet is high.



Limno-Terrestrial Tardigrades.  Tardigrades that have evolved to survive in limno-terrestrial habitats can either be gonochoristic, hermaphroditic, or parthenogenic. Adaptation to limno-terrestrial habitats has allowed tardigrades in isolated and constantly changing environments to survive harsh conditions via hermaphroditism or parthenogenesis (Bertolani 2001).

Gonochorism is very common in freshwater and semi-terrestrial tardigrades with a higher presence in eutardigrades than heterotardigrades (Bertolani 1987). The freshwater males are slightly smaller than females and are semelparous (Bertolani 2001). Before the males mate, all the sperm in their testes must mature. More than one male may attempt to copulate with the female, using their modified front claws to attach to her (Bertolani 2001). The male will die after mating. Females are iteroparous and can lay several eggs during her lifespan (Bertolani 2001). In contrast, in some moss and leaf litter species, males are usually the same size as the females and possess sperm of all stages in their testes (Bertolani 2001). They can copulate several times throughout their life.



     Males exist in populations when there is sufficient opportunity for contact with females (Glime 2007), otherwise reproduction via hermaphroditism or parthenogenesis exerts an advantage – they allow one individual to successfully colonize a new environment. Hermaphroditism is a derived trait frequently found in most families of eutardigrades including Hypsibius, but it is sporadic and less common than gonochorism (Bertolani 1987). Hermaphroditic tardigrades self-fertilize and are found in “low-density” environments (Ghiselin 1969). This form of reproduction has higher fitness costs associated with it, however, only one tardigrade is needed to reproduce.



     After colonizing a site, parthenogenic species reproduce asexually and can create “cryptic species” through drift, a bottleneck, and/or microselection (Glime 2007). Thelytoky is the only type of parthenogenesis found in tardigrades (Bertolani 2001). Parthenogenic species are commonly found on mosses and lichens, leaf litter, soil, and in freshwater (Glime 2007). On a wider distribution, males are absent in Europe, Africa, and America; they hardly occur in Asia. Males are only frequent in Antarctica and Australia (Bertolani 2001). Parthenogenesis and self-fertilization never occur together in tardigrade species, therefore, one can infer that self-fertilization will only evolve where parthenogenesis is absent (Glime 2007).



Reproductive Anatomy.  In both males and females, one gonad is found just above the intestine (Barnes 1982). The gonad can change size depending on the developmental state of the individual (Bertolani 1987). The midgut and female gonad alternate in size in many females and allows them to maintain their small size. However, during molting, the gonad grows until it reaches almost the entire size of the body (Bertolani 1987).



     Most species have external fertilization with sperm being deposited on the eggs inside of the shed cuticle (Bertolani 2001). Other species mate before the female sheds her cuticle. Traumatic insemination, where males penetrate the cuticle of the female and deposit sperm into her hemocoel, is seen in at least one species (Biodiversity Institute 2008).
Eggs. Females may lay one to thirty eggs at a time (Biodiversity Institute 2008, Gagyi-Palffy and Stoian 2011). The number of eggs laid depends on the species and nutritional status of the female (Glime 2007). In most cases, the eggs develop in the exuvia (shed exoskeleton) until hatching (Glime 2007), but some attach them to the nearby substrate (Barnes 1982) Most aquatic species either leave the eggs in their shed cuticle or glue the eggs to the empty exoskeletons of cladocerans, ostracods, or insects (Biodiversity Institute 2008). Some tardigrades have been shown to use moss capsules to lay their eggs in.

     The structure of the eggs varies with the mode of egg storage. Eggs laid in the shed exoskeleton are smooth, while free eggs are ornamented with pores and reticulations; some resemble pollen grains (Bertolani 2001). However, all eggs possess a defensive structure, the ability for gas exchange, and a water reservoir to keep the egg from drying out (Kinchin 1994). Tardigrade eggs take five to fourteen days to hatch (Figure 7). Occasionally, they make take up to forty days to hatch, but even ninety days is uncommon if the eggs go into a cryptobiotic state (Glime 2007). Hatched tardigrades may be smaller than 0.05 millimeters. Metamorphosis does not occur in tardigrades; therefore hatched tardigrades resemble adults (Gagyi-Palffy and Stoian 2011, Pechenik 2010). Temperature and body size have been shown to affect the growth and development of tardigrades (Glime 2007).



Conclusion



     Tardigrades are widely dispersed in the environment, yet zoologists have largely ignored them. Their relationship to other taxa and ability to live and survive in a multitude of environments has driven much of the recent research of tardigrades. They have become a model organism next to Drosophila and C. elegans and future questions involving the world of the tardigrades wait to be investigated.