Trehalose and anhydrobiosis in tardigrades--evidence for divergence in responses to dehydration

To withstand desiccation, many invertebrates such as rotifers, nematodes and tardigrades enter a state known as anhydrobiosis, which is thought to require accumulation of compatible osmolytes, such as the non-reducing disaccharide trehalose to protect against dehydration damage. The trehalose levels of eight tardigrade species comprising Heterotardigrada and Eutardigrada were observed in five different states of hydration and dehydration. Although many species accumulate trehalose during dehydration, the data revealed significant differences between the species. Although trehalose accumulation was found in species of the order Parachela (Eutardigrada), it was not possible to detect any trehalose in the species Milnesium tardigradum and no change in the trehalose level has been observed in any species of Heterotardigrada so far investigated. These results expand our current understanding of anhydrobiosis in tardigrades and, for the first time, demonstrate the accumulation of trehalose in developing tardigrade embryos, which have been shown to have a high level of desiccation tolerance.     

Desiccation tolerance of the tardigrade Milnesium tardigradum collected in Sapporo, Japan, and Bogor, Indonesia

A tardigrade Milnesium tardigradum showed anhydrobiotic capacity, in which the desiccation tolerance, given by the mean survival rate under desiccation at different relative humidity levels, was significantly higher in the Sapporo (Japan) population than that in the Bogor (Indonesia) population. Accordingly, the surviving tardigrades took a significantly longer time for revival in Bogor than those in Sapporo. The higher tolerance of the Sapporo population is thought to be related to the low relative humidity and low temperature such that the animals experience 41% RH in May and often -10 degrees C or lower in winter.     

Comparative studies on storage cells in tardigrades during starvation and anhydrobiosis

The impact of starvation and anhydrobiosis on the number and size of the storage cells in the tardigrade species Milnesium tardigradum, Paramacrobiotus tonollii and Macrobiotus sapiens was investigated to gain more insight on the energetic side of anhydrobiosis. Storage cells are free floating cells within the body cavity of tardigrades and are presumed to store and release energy in form of glycogen, protein and fat to maintain a constant nutrient regime for the other tissues. The body size of the animals was not correlated with the size of the storage cells, however, M. tardigradum the largest species analysed also had the largest storage cells. A reduction in the size of the storage cells is apparent in all three species after seven days of starvation. A seven-day period of anhydrobiosis leads to a decrease in cell size in M. tardigradum but not in P. tonollii and M. sapiens. Although M. sapiens was raised on green algae, and M. tardigradum and P. tonollii were fed with rotifers and nematodes this difference in nourishment was not reflected in the response of the storage cells to anhydrobiosis.     

New tardigrade records for the Baltic states with a description of Minibiotus formosus sp. n. (Eutardigrada,Macrobiotidae)

In sixteen moss, lichen and mixed (moss/lichen) samples, collected from Estonia, Latvia and Lithuania, 291 specimens, 48 simplexes, including one exuvium with 6 eggs, and 8 free-laid eggs of eutardigrades were found. In total, 17 species, together with one new to science, were identified (all are new records for the Baltic states): Astatumen bartosi, Diphascon (Adropion) prorsirostre, D. (Diphascon) bullatum, D. (D.) pingue pingue, D. (D.) recamieri, D. (D.) rugosum, Hypsibius convergens, H. dujardini, H. cf. scabropygus, Isohypsibius ronsisvallei, I. sattleri, Macrobiotus harmsworthi harmsworthi, M. hufelandi hufelandi, Milnesium asiaticum, Milnesium tardigradum tardigradum, Minibiotus formosus sp. n. and Paramacrobiotus richtersi. The new species is most similar to Minibiotus gumersindoi, but differs from it mainly by the presence of two types of cuticular pores, the absence of a triangular or pentagonal arrangement of pores above a single large pore on legs, the presence of granulation on all legs and a different macroplacoid length sequence. In this paper we also provide photographs and morphometrics of H. cf. scabropygus.     

Anhydrobiosis in tardigrades and its effects on longevity traits

Living in harsh and variable environments that are prone to periodic desiccation, tardigrades exhibit remarkable tolerance against physical extremes through a state known as anhydrobiosis. To study the effect of this state on the longevity and hence the lifecycle in the taxon Tardigrada for the first time, we exposed a tardigrade species, Milnesium tardigradum , to alternating periods of drying and active feeding periods in a hydrated state. Compared with a hydrated control, the periodically dried animals showed a similar longevity, indicating that the time spent in anhydrobiosis was ignored by the internal clock. Thus, desiccation can produce a time shift in the age of tardigrades similar to the model described for rotifers that has been termed 'Sleeping Beauty'.     

Appearance of males in a thelytokous strain of Milnesium cf. tardigradum (Tardigrada)

Tardigrades are generally gonochoristic. Many moss-dwelling species propagate by parthenogenesis, but heterogony has not yet been found. Milnesium tardigradum, a carnivorous tardigrade, also has both sexes, but males are usually rare and many populations appear to have only parthenogenetic reproduction. Since 2000, I have maintained a thelytokous strain of Milnesium cf. tardigradum that originated from one female. Individuals of this strain were thought to be all females, but here I report that males have emerged in this strain at a very low frequency. This is the first report of the appearance of males in parthenogenetic tardigrades. On the first pair of legs of some individuals, I observed the modified claws characteristic of males of this species. It is unknown whether these males can actually function in sexual reproduction; however, they might allow some possibility of genetic exchange among clonal populations. No environmental factors that generate males were determined.     

Freeze-fracture studies on tardigrade cuticle

The cuticles of the heterotardigrade Echiniscus testudo and the eutardigrades Macrobiotus hufelandi and Milnesium tardigradum have been studied using freeze-fracture technique. Most of the layers seen in conventional TEM micrographs can be visualized. There is no clear evidence that the trilaminar components of the cuticle such as the outer epicuticle and the tripartite layer separating epi- and intracuticle or procuticle (whose membranous origin has been suggested by previous authors) fracture like a lipid bilayer. Microfibres not resolved or only poorly resolved by TEM can be recognized in the procuticle of all three species. Obviously their visualization depends upon the fracture angle. In Echiniscus testudo and Milnesium tardigradum the intracuticle or at least parts of it show a wavy arrangement of microfibres. Parts of the ventral intracuticle of E. testudo fracture in an obviously non-random pattern revealing distinct sublayers.     

Muscular architecture of <Emphasis Type="Italic">Milnesium tardigradum </Emphasis>and <Emphasis Type="Italic">Hypsibius </Emphasis>sp. (Eutardigrada,Tardigrada) with some data on <Emphasis Type="Italic">Ramazottius oberhaeuseri</Emphasis>

The architecture of the musculature of the eutardigrade species Milnesium tardigradum Doyère, 1840, Hypsibius sp. and Ramazzottius oberhaeuseri (Doyère in Ann Sci Nat Zool Sér 2(14):269–369, 1840) is investigated by phalloidin staining and confocal laser scanning microscopy. There are methodological problems in staining eutardigrades due to physiological alterations under stress (anhydrobiosis) and due to penetration problems of the cuticle. It is helpful to fix specimens in the state of asphyxy, where animals are stretched following an oxygen shortage in their environment. The musculatures of all three species correspond in their general architecture, but differ in detail, such as in the number of muscles. All muscles are isolated muscle strands. There are on each body side two dorsal and one ventral muscle strands, in addition to a system of dorsoventral, lateral and lateroventral muscles. Seven median ventral attachment points give rise to dorsoventral, ventrolateral and appendage muscles. The appendages receive several muscles originating dorsally and ventrally. The number of muscles and the arrangement differ in each appendage. The fourth appendage shows the greatest differences with a far smaller number of muscles compared to other species. The musculature shows comparably few strict segmental patterns, for example, the musculature of each appendage differs from the other ones. By comparison with literature data on the same species and data of Macrobiotus hufelandi it can be shown that eutardigrades have a roughly comparable muscular architecture, but that there are several differences in detail. Dedicated to Professor Westheide on the occasion of his 70th birthday.     

Form and function of the feeding apparatus in Eutardigrada (Tardigrada)

Tardigrade feeding apparatus is a complex structure with considerable taxonomic significance that can be schematically divided into four parts: buccal ring, buccal tube, stylet system, and pharynx. We analyzed the fine morphology and the tridimensional organization of the tardigrade buccal?Cpharyngeal apparatus in order to clarify the relationships between form and function and to identify new characters for systematic and phylogenetic studies. We conducted a comparative analysis of the cuticular structures of the buccal?Cpharyngeal apparatuses of twelve eutardigrade species, integrating data obtained by SEM and LM observations. Morphological diversity was observed and new cuticular structures such as the stylet coat of the stylet system were identified. The synthesis of the buccal?Cpharyngeal apparatus during molting was also analyzed obtaining a clear developmental sequence of its resynthesis. These findings lead us to redefine the previous interpretations of the functioning mechanisms of the buccal?Cpharyngeal apparatus and provide a more specific relationship between tardigrade diet and the anatomy of their feeding apparatuses. In addition, the detection by energy-dispersive X-ray spectroscopy of calcium in the stylets, buccal tube, and placoids of eutardigrade species (i.e., Milnesium tardigradum, Paramacrobiotus richtersi) indicates that CaCO₃ incrustations are not an exclusive feature of heterotardigrades and lead to suppose that this trait was present in the ancestors of both classes.     

Proteomic Analysis of Tardigrades: Towards a Better Understanding of Molecular Mechanisms by Anhydrobiotic Organisms

Background

Tardigrades are small, multicellular invertebrates which are able to survive times of unfavourable environmental conditions using their well-known capability to undergo cryptobiosis at any stage of their life cycle. Milnesium tardigradum has become a powerful model system for the analysis of cryptobiosis. While some genetic information is already available for Milnesium tardigradum the proteome is still to be discovered.

Principal Findings

Here we present to the best of our knowledge the first comprehensive study of Milnesium tardigradum on the protein level. To establish a proteome reference map we developed optimized protocols for protein extraction from tardigrades in the active state and for separation of proteins by high resolution two-dimensional gel electrophoresis. Since only limited sequence information of M. tardigradum on the genome and gene expression level is available to date in public databases we initiated in parallel a tardigrade EST sequencing project to allow for protein identification by electrospray ionization tandem mass spectrometry. 271 out of 606 analyzed protein spots could be identified by searching against the publicly available NCBInr database as well as our newly established tardigrade protein database corresponding to 144 unique proteins. Another 150 spots could be identified in the tardigrade clustered EST database corresponding to 36 unique contigs and ESTs. Proteins with annotated function were further categorized in more detail by their molecular function, biological process and cellular component. For the proteins of unknown function more information could be obtained by performing a protein domain annotation analysis. Our results include proteins like protein member of different heat shock protein families and LEA group 3, which might play important roles in surviving extreme conditions.

Conclusions

The proteome reference map of Milnesium tardigradum provides the basis for further studies in order to identify and characterize the biochemical mechanisms of tolerance to extreme desiccation. The optimized proteomics workflow will enable application of sensitive quantification techniques to detect differences in protein expression, which are characteristic of the active and anhydrobiotic states of tardigrades.     

Desiccation tolerance in embryonic stages of the tardigrade

Desiccation tolerance commonly found among tardigrades allows them to cope with temporal variation of available water. Although the long-term survival of adults has been demonstrated in several species, desiccation tolerance of eggs and embryos is less well studied, however it is an important aspect from an ecological and evolutionary point of view. For the first time we evaluated the desiccation tolerance and subsequent hatching success of five different developmental stages of the tardigrade species Milnesium tardigradum , when rehydrated following drying at eight different humidity levels (10, 20, 31, 40, 54, 59, 72, 81%). Humidity level and developmental stage are significant factors in determining successful hatch rates. The results showed that the less developed stages were quite sensitive to desiccation. Low humidity levels during the first 3 days of development lead to a decrease in hatch rates following rehydration. Later developmental stages showed higher hatch rates than embryos dried at earlier stages. However, fast drying at low humidity levels resulted in delayed development and lower hatch rates following rehydration. In general, further developed embryos exhibit a better survival capacity compared with younger stages.     

Seasonal and Altitudinal Variation in the Distribution and Abundance of Tardigrada on Dugger Mountain, Alabama

A seasonal survey of the distribution of terrestrial tardigrades on Dugger Mountain, Alabama, was conducted during the time period from April 1997 through April 1998. Cryptogams from five trees (Quercus alba), three on north-facing slopes and two on south-facing slopes, were sampled seasonally at three stations (645 m, 410 m, 183 m) along an unnamed tributary of the South Fork of Terrapin Creek. Trees were chosen based on their location outside the riparian zone. Tardigrades were extracted from the samples, mounted individually in Hoyer's medium, and identified to species with phase microscopy. Seasonal and altitudinal variations in the distribution of the populations on the north- and south-facing slopes were determined. Present on Dugger Mountain were tardigrades belonging to 12 species (Macrobiotus cf. areolatus/tonollii, Macrobiotus cf. echinogenitus, Macrobiotus islandicus, Macrobiotus richtersi, Minibiotus intermedius, Milnesium tardigradum, Diphascon pingue, Hypsibius pallidus, Echiniscus cf. arctomys, Echiniscus virginicus, Pseudechiniscus ramazzottii, and Pseudechiniscus suillus). Due to the small numbers of individuals of each species, the total numbers of tardigrades of all species were pooled. There were no significant differences in the mean number of species or the mean number of all tardigrades per sample at each station (altitude). However, seasonal differences in both abundance and number of species were detected in pooled samples due to the high numbers collected in spring 1997.     

A new species of Ramazzottius (Tardigrada, Hypsibiidae) in a rain gutter sediment from England

Five species of tardigrades were extracted from a sample of sediment from a rain gutter. Four of these, the heterotardigrade Echiniscus testudo and the eutardigrades Milnesium tardigradum, Ramazzottius oberhaeuseri , and probably Macrobiotus cf. sandrae are typical of xerophilous habitats. The fifth species, Ramazzottius varieornatus sp. nov. , is new to science, and it can be readily distinguished from the others of the same genus by egg shell morphology, placoids and the type of cuticle.     

Population density and species composition of moss-living tardigrades in a boreo-nemoral forest

This study investigates for the first time the tardigrade fauna in a variety of different mosses from a coniferous forest and an adjacent clear-cut area in southern Sweden. Tardigrades were found in a majority of the samples. Sixteen species were recorded, of which the cosmopolitan species Macrobiotus hufelandi was the far most common. Some mosses, particularly species with "wefts" growth form, contained more tardigrades than other mosses, indicating that growth form may have an impact on tardigrade abundance. Mosses of the same species collected from a forest and from a clear-cut, respectively, did not show a general trend in the overall abundance of tardigrades, but the forest tended to contain more species. Five species of tardigrades ( Murrayon dianae, Isohypsibius sattleri, Platicrista angustata, Diphascon belgicae and Diphascon pingue ) never previously reported from Sweden were recorded.     

Milnesium berladnicorum sp. n. (Eutardigrada,Apochela, Milnesiidae), a new species of water bear from Romania

In a lichen sample collected from a tree in Bârlad town (Vaslui County, Romania), a new tardigrade species belonging to the genus Milnesium (granulatum group) was found. Milnesium berladnicorum sp. n. is most similar (in the type of dorsal sculpture) to Milnesium beasleyi Kaczmarek et al., 2012 but differs from it mainly by having a different claw configuration and some morphometric characters. Additionally, the new species differs from other congeners of the granulatum group by the different type of dorsal sculpture, claw configuration and some morphometric characters.     

Geonemy,ecology, reproductive biology and morphology of the tardigrade <Emphasis Type="Italic">Hypsibius zetlandicus</Emphasis> (Eutardigrada: Hypsibiidae) with erection of <Emphasis Type="Italic">Borealibius</Emphasis> gen. n.

The morphology, biology and geographic distribution of Hypsibius zetlandicus (Murray 1907) are considered. Scanning electron microscopy (SEM) and/or light microscopy (LM) analyses have been carried out on H. zetlandicus and the type species of Hypsibius: Hypsibius dujardini (Doyère 1840), with particular emphasis on the buccal–pharyngeal apparatuses. Some unusual characteristics of this apparatus in H. zetlandicus lead us to the erection of the new genus Borealibius, to which H. zetlandicus (Borealibius zetlandicus comb. n.) is transferred. In the light of new discoveries of this species from polar, sub-polar and alpine regions, and based on the available bibliographic references, we hypothesize a boreo–alpine distribution for this species. The presence of traits that are unusual and rare (in other tardigrade species) have been observed whilst analyzing the reproductive biology and ecology of B. zetlandicus (i.e., the presence of hermaphroditism, parental care and the colonization of very different substrates).     

Vergleichende Untersuchungen am Integument von Hetero- und Eutardigraden

Zusammenfassung Licht- und elektronenmikroskopische Untersuchungen am Integument vonEchiniscus testudo (Heterotardigrada),Milnesium tardigradum, Hypsibius oberhaeuseri undMacrobiotus hufelandi (Eutardigrada) lassen acht, mindestens jedoch sechs Schichten erkennen, die in Anlehnung an Baccetti und Rosati (1971) benannt werden, wobei eine Homologie beiE. testudo nicht sicher ist.BeiE. testudo bildet die innere sklerotisierte Epicuticula ein Hohlraumsystem, dessen Wände von ca. 150 Å breiten Kanälen durchzogen werden. Die dorsalen Einsenkungen der Panzerplatten besitzen einen zentralen 500 Å breiten Kanal. Ventral ist die äußere Epicuticula mehrfach geschichtet. Ein darunterliegender Röhrchensaum und die weitgehend reduzierte innere Epicuticula lassen andere Permeabilitätseigenschaften vermuten. Die Intracuticula zeigt vertikale Filamente. Wachsschicht und innere Lage der Intracuticula sind morphologisch nicht eindeutig zu identifizieren. Die einschichtige Epidermis ist reich an Lipidtropfen, freien Ribosomen und granulärem endoplasmatischem Reticulum. Manche Zellen sind kontrastreicher.Besondere Cuticula-Bildungen der drei Eutardigraden sind die epicuticulären Einsenkungen vonM. hufelandi, in deren Bereich die innere Epicuticula fehlt, die dorsale und laterale Skulpturierung vonH. oberhaeuseri, die eine Bildung der Epicuticula ist, und die epicuticulären Kanäle vonM. tardigradum. Die Proticula aller untersuchten Tardigraden-Arten besitzt keine Porenkanäle.Die Epidermis vonM. hufelandi besteht aus einem einschichtigen Epithel. Seine Zellen enthalten zahlreiche membranbegrenzte osmiophile Einschlüsse, die die Färbung der Tiere bedingen. Einzelne Zellen fallen durch ihren hohen Kontrast und zahlreichere Organellen auf.Die Cuticula der Eutardigraden zeigt in allen Fällen eine morphologisch vergelichbare Schichtenfolge. Die Cuticula vonE. testudo, die Besonderheiten aufweist, kann jedoch aufgrund charakteristischer Merkmale (z. B. die äußere Lage der Intracuticula) vielleicht in den Vergleich einbezogen werden.

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Comparative studies on the integument of Hetero- and Eutardigrada

Summary The structure of the integument ofEchiniscus testudo (Heterotardigrada),Milnesium tardigradum, Hypsibius oberhaeuseri andMacrobiotus hufelandi (Eutardigrada) was studied by light and electron microscopy. In all species the cuticle consists of 6–8 layers and can adequately be described following the terminology of Baccetti and Rosati (1971). This perhaps does not apply toE. testudo.InE. testudo the inner sclerotized epicuticle reveals a labyrinthine structure due to lacunae with numerous channels of about 150 Å in diameter. The central canals in the depressions of the dorsal plates have a diameter of 500 Å. The outer epicuticle of the ventral cuticle is formed by several layers. Basally there is a tubular layer of channels and an almost reduced epicuticle. Thus the permeability of the cuticle is supposed to be altered. In the intracuticle vertically oriented filaments occur. The inner layer of the intracuticle and the wax-layer cannot be properly distinguished. The one-layered epidermis contains many lipid droplets, ribosomes and rough ER. Some of the cells are rather electron-dense.Peculiar cuticular structures inM. hufelandi appear as cup-shaped depressions of the epicuticle, and here the inner epicuticle is absent. A similarly typical feature is the structured and sculptured dorsal and lateral epicuticle ofH. oberhaeuseri and the channels of the epicuticle inM. tardigradum. In all species investigated there are no pore canals in the procuticle.InM. hufelandi there is a single layer of epidermal cells, which contain many osmiophilic grana ensheathed by membranes. These grana cause the colour of the animal. Some of the cells contain more organelles and ribosomes and show a strong contrast.In spite of some cuticular peculiarities inE. testudo, perhaps this Heterotardigrade species fits into the general pattern of cuticle morphology of Tardigrada.

Veränderter Teil einer Dissertation des Fachbereichs Biologie der Universität Münster. Die Arbeit wurde von Herrn Prof. Dr. R. Altevogt angeregt.