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Concept: Cryptobiosis

368

Long-term survival has been one of the most studied of the extraordinary physiological characteristics of cryptobiosis in micrometazoans such as nematodes, tardigrades and rotifers. In the available studies of long-term survival of micrometazoans, instances of survival have been the primary observation, and recovery conditions of animals or subsequent reproduction are generally not reported. We therefore documented recovery conditions and reproduction immediately following revival of tardigrades retrieved from a frozen moss sample collected in Antarctica in 1983 and stored at -20°C for 30.5 years. We recorded recovery of two individuals and development of a separate egg of the Antarctic tardigrade, Acutuncus antarcticus, providing the longest records of survival for tardigrades as animals or eggs. One of the two resuscitated individuals and the hatchling successfully reproduced repeatedly after their recovery from long-term cryptobiosis. This considerable extension of the known length of long-term survival of tardigrades recorded in our study is interpreted as being associated with the minimum oxidative damage likely to have resulted from storage under stable frozen conditions. The long recovery times of the revived tardigrades observed is suggestive of the requirement for repair of damage accrued over 30 years of cryptobiosis. Further more detailed studies will improve understanding of mechanisms and conditions underlying the long-term survival of cryptobiotic organisms.

Concepts: Arthropod, Cultural studies, Tardigrade, Trehalose, Antarctica, Cryptobiosis, Antarctic Circle, Antarctic

179

Tardigrades are able to tolerate almost complete dehydration by reversibly switching to an ametabolic state. This ability is called anhydrobiosis. In the anhydrobiotic state, tardigrades can withstand various extreme environments including space, but their molecular basis remains largely unknown. Late embryogenesis abundant (LEA) proteins are heat-soluble proteins and can prevent protein-aggregation in dehydrated conditions in other anhydrobiotic organisms, but their relevance to tardigrade anhydrobiosis is not clarified. In this study, we focused on the heat-soluble property characteristic of LEA proteins and conducted heat-soluble proteomics using an anhydrobiotic tardigrade. Our heat-soluble proteomics identified five abundant heat-soluble proteins. All of them showed no sequence similarity with LEA proteins and formed two novel protein families with distinct subcellular localizations. We named them Cytoplasmic Abundant Heat Soluble (CAHS) and Secretory Abundant Heat Soluble (SAHS) protein families, according to their localization. Both protein families were conserved among tardigrades, but not found in other phyla. Although CAHS protein was intrinsically unstructured and SAHS protein was rich in β-structure in the hydrated condition, proteins in both families changed their conformation to an α-helical structure in water-deficient conditions as LEA proteins do. Two conserved repeats of 19-mer motifs in CAHS proteins were capable to form amphiphilic stripes in α-helices, suggesting their roles as molecular shield in water-deficient condition, though charge distribution pattern in α-helices were different between CAHS and LEA proteins. Tardigrades might have evolved novel protein families with a heat-soluble property and this study revealed a novel repertoire of major heat-soluble proteins in these anhydrobiotic animals.

Concepts: DNA, Proteins, Protein, Cell, Bioinformatics, Molecular biology, Tardigrade, Cryptobiosis

151

Tardigrades are ubiquitous microscopic animals that play an important role in the study of metazoan phylogeny. Most terrestrial tardigrades can withstand extreme environments by entering an ametabolic desiccated state termed anhydrobiosis. Due to their small size and the non-axenic nature of laboratory cultures, molecular studies of tardigrades are prone to contamination. To minimize the possibility of microbial contaminations and to obtain high-quality genomic information, we have developed an ultra-low input library sequencing protocol to enable the genome sequencing of a single tardigrade Hypsibius dujardini individual. Here, we describe the details of our sequencing data and the ultra-low input library preparation methodologies.

Concepts: DNA, Genetics, Molecular biology, Organism, Animal, Tardigrade, Trehalose, Cryptobiosis

28

Tardigrades, commonly known as water bears, are tiny invertebrates capable of suspending their metabolism when exposed to adverse environmental conditions. While this process, called tunning, has been heavily studied, research on behavioral conditioning of tardigrades is practically nonexistent. Even at the basic associative level, tardigrade learning remains unexplored. In this study, Hypsibius dujardini tardigrades were classically conditioned using a starvation regimen to induce positive chemotaxis to a conditioned stimulus. Two different training regimens were used: (1) massed training, which involved one block of starvation and subsequent exposure to the conditioned stimulus (acetone) with algal food and (2) spaced training, in which seven such training blocks were conducted. After training under both regimens, tardigrades were tested on preference between acetone and EtOH (the control) until behavior returned to naïve levels. After establishing the learning decay of both training regimens, tardigrades were then tunned before and during testing to determine the effects of suspended metabolism on associative memory. Results showed that massed training resulted in learning decay of roughly 5 h, whereas spaced training yielded behavioral change persisting up to 35 h. Suspended animation interfered only with the short-term memory, causing tardigrades to behave like naïve specimens after training. These findings demonstrate that (a) tardigrades can form associative memories as a result of Pavlovian conditioning and that (b) short-term learning requires ongoing neuronal processes (as opposed to CREB-dependent structural changes) to produce behavioral change.

Concepts: Psychology, Arthropod, Memory, Behaviorism, Classical conditioning, Tardigrade, Ivan Pavlov, Cryptobiosis

28

Many species of tardigrades are known to tolerate extreme environmental stress, yet detailed knowledge of the mechanisms underlying the remarkable adaptations of tardigrades is still lacking, as are answers to many questions regarding their basic biology. Here, we present data on the inorganic ion composition and total osmotic concentration of five different species of tardigrades (Echiniscus testudo, Milnesium tardigradum, Richtersius coronifer, Macrobiotus cf. hufelandi and Halobiotus crispae) using high-performance liquid chromatography and nanoliter osmometry. Quantification of the ionic content indicates that Na(+) and Cl(-) are the principle inorganic ions in tardigrade fluids, albeit other ions, i.e. K(+), NH(4)(+), Ca(2+), Mg(2+), F(-), SO(4)(2-) and PO(4)(3-) were also detected. In limno-terrestrial tardigrades, the respective ions are concentrated by a large factor compared to that of the external medium (Na(+), ×70-800; K(+), ×20-90; Ca(2+) and Mg(2+), ×30-200; F(-), ×160-1040, Cl(-), ×20-50; PO(4)(3-), ×700-2800; SO(4)(2-), ×30-150). In contrast, in the marine species H. crispae Na(+), Cl(-) and SO(4)(2-) are almost in ionic equilibrium with (brackish) salt water, while K(+), Ca(2+), Mg(2+) and F(-) are only slightly concentrated (×2-10). An anion deficit of ~120 mEq 1(-1) in M. tardigradum and H. crispae indicates the presence of unidentified ionic components in these species. Body fluid osmolality ranges from 361±49 mOsm kg(-1) in R. coronifer to 961±43 mOsm kg(-1) in H. crispae. Concentrations of most inorganic ions are largely identical between active and dehydrated groups of R. coronifer, suggesting that this tardigrade does not lose large quantities of inorganic ions during dehydration. The large osmotic and ionic gradients maintained by both limno-terrestrial and marine species are indicative of a powerful ion-retentive mechanism in Tardigrada. Moreover, our data indicate that cryptobiotic tardigrades contain a large fraction of unidentified organic osmolytes, the identification of which is expected to provide increased insight into the phenomenon of cryptobiosis.

Concepts: Concentration, Chromatography, High performance liquid chromatography, Analytical chemistry, Ion, Tardigrade, Trehalose, Cryptobiosis

28

Tardigrades are microscopic metazoans which are able to survive extreme physical and chemical conditions by entering a stress tolerant state called cryptobiosis. At present, the molecular mechanisms behind cryptobiosis are still poorly understood. We show that surface enhanced Raman scattering supported by plasmonic gold nanoparticles can measure molecular constituents and their local distribution in live tardigrades. Surface enhanced Raman signatures allow to differentiate between two species and indicate molecular structural differences between tardigrades in water and in a dry state. This opens new avenues for exploring cryptobiosis by studying molecular changes in live cryptobiotic organisms. (© 2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim).

Concepts: DNA, Molecule, Scattering, Gold, Raman scattering, Tardigrade, Trehalose, Cryptobiosis

9

Life unfolds within a framework of constraining abiotic factors, yet some organisms are adapted to handle large fluctuations in physical and chemical parameters. Tardigrades are microscopic ecdysozoans well known for their ability to endure hostile conditions, such as complete desiccation - a phenomenon called anhydrobiosis. During dehydration, anhydrobiotic animals undergo a series of anatomical changes. Whether this reorganization is an essential regulated event mediated by active controlled processes, or merely a passive result of the dehydration process, has not been clearly determined. Here, we investigate parameters pivotal to the formation of the so-called “tun”, a state that in tardigrades and rotifers marks the entrance into anhydrobiosis. Estimation of body volume in the eutardigrade Richtersius coronifer reveals an 87 % reduction in volume from the hydrated active state to the dehydrated tun state, underlining the structural stress associated with entering anhydrobiosis. Survival experiments with pharmacological inhibitors of mitochondrial energy production and muscle contractions show that i) mitochondrial energy production is a prerequisite for surviving desiccation, ii) uncoupling the mitochondria abolishes tun formation, and iii) inhibiting the musculature impairs the ability to form viable tuns. We moreover provide a comparative analysis of the structural changes involved in tun formation, using a combination of cytochemistry, confocal laser scanning microscopy and 3D reconstructions as well as scanning electron microscopy. Our data reveal that the musculature mediates a structural reorganization vital for anhydrobiotic survival, and furthermore that maintaining structural integrity is essential for resumption of life following rehydration.

Concepts: Electron, Metabolism, Mitochondrion, Muscle contraction, Dehydration, Scanning electron microscope, Tardigrade, Cryptobiosis

1

Many tardigrade species are capable of anhydrobiosis; however, mechanisms underlying their extreme desiccation resistance remain elusive. This study attempts to quantify the anhydrobiotic transcriptome of the limno-terrestrial tardigrade Milnesium tardigradum.

Concepts: Tardigrade, RNA-Seq, Cryptobiosis

0

Earth’s geomagnetic field has undergone critical changes in the past. Studies on the influence of the magnetic field on Earth’s organisms are crucial for the understanding of evolution of life on Earth and astrobiological considerations. Numerous studies conducted both on plants and animals confirmed the significant influence of the geomagnetic field on the metabolism of living organisms. Water bears (Tardigrada), which are a mong the most resistant animals due to their cryptobiotic abilities, show significant resistance to a number of environmental stressors, but the influence of the geomagnetic field on their fitness has not been addressed before. In our studies, we used eutardigrade Hypsibius dujardini to analyse whether isolation from the geomagnetic field had an effect on mortality. We found that Hypsibius dujardini specimens demonstrated relatively high mortality during anhydrobiosis, also in control groups exposed to the normal geomagnetic field. Moreover, similar mortality was observed in anhydrobiotic specimens isolated from the geomagnetic field. However, a significant difference was noted between tardigrade survival and the moment of their isolation from the geomagnetic field. In particular, tardigrade mortality substantially increased in absence of a magnetic field during the process of entering anhydrobiosis and returning to active life. Our results suggest that these processes rely on complex metabolic processes that are critically influenced by the geomagnetic field.

Concepts: Magnetic field, Evolution, Metabolism, Biology, Organism, Life, Tardigrade, Cryptobiosis

0

Some organisms can survive extreme desiccation caused by hypertonic osmotic pressure by entering a state of suspended animation known as osmobiosis. The free-living mycophagous nematode Aphelenchoides besseyi can be induced to enter osmobiosis by soaking in osmolytes. It is assumed that sugars (in particular trehalose) are instrumental for survival under environmental stress. In A. besseyi two putative trehalose-6-phosphate synthase genes (TPS) encode enzymes catalyzing trehalose synthesis and a putative trehalase genes (TRE) encodes enzymes that catalyze hydrolysis of trehalose were identified and then characterized based on its transcriptome. RT-qPCR analyses showed that each of these genes is expressed as mRNA when A. besseyi is entering in, during and recovering from osmobiosis, but only for certain periods. The changes of TRE activity were consisted with the transcript level changes of TRE gene and the trehalose level declined a lot at certain periods when the nematodes were in osmobiosis as well as recovering from osmobiosis. Which suggested that the hydrolysis of threhalose is essential. The feeding method of RNA interference (RNAi) was used to temporarily knock down the expression of each of the TPS and TRE genes. No obviously different phenotype was observed from any of the genes silenced individually or simultaneously but the survival under hypertonic osmotic pressure reduced significantly and the recovery was delayed. These results indicated that trehalose metabolism genes should play a role in osmobiosis regulation and also function within a restricted time frame.

Concepts: Protein, Gene, Gene expression, Bacteria, Enzyme, Virus, RNA, Cryptobiosis