Hypsibius Dujardini Research Articles

Using Electron Paramagnetic Resonance Spectroscopy to Determine the Role of Free‐Radical Protection in Tardigrade Survival Mechanisms

As aeronautic technology advances, so does the possibility of long‐term space travel and exploration. However, in order to make significant strides, it is first necessary to determine how to survive the antagonistic space environment, including temperature extremes, UVC radiation, and vacuum pressures, among others. Tardigrades are microscopic invertebrates that possess the ability to adapt to extreme environments through the formation of a tun, a dormant structure resulting from the downregulation of metabolism. This ability to survive under extreme conditions, including arctic temperatures, extreme heat, and a pure vacuum, makes tardigrades a useful model organism for space survival. By increasing our understanding of their survival mechanisms on a fundamental level, we may be able to generate technologies that protect humans in space from diseases caused by ROS and the damaging effects of oxidative stress.Previous research has examined the physiological response of tardigrades subjected to freezes and irradiation that model outer Earth conditions. In these studies, tardigrades were shown to enter tun formation through cryptobiosis, especially in response to physical stressors causing desiccation. Another study assessed tardigrades’ intergalactic survival capability by sending a culture into the vacuum of space, after which the cultures showed no loss of viability. We hypothesize that tardigrades have a specific, protective mechanism to respond to oxidative stress through the increased expression of superoxide dismutases (SODs) to break down potentially harmful ROS, which is supported by previous studies showing both the formation of superoxide radicals as well as an increase in SODs in desiccated animals.In this study, X‐band continuous wave electron paramagnetic resonance (EPR) spectroscopy was used to measure the presence of superoxide radicals in the tardigrade Hypsibius exemplaris before and after exposure to environmental stressors. Tardigrades were subjected to a combination of UVA and UVB irradiation before the addition of a spin probe, and the resulting EPR spectrum was compared to a nonirradiated H. exemplaris control. Results show that the tardigrades generated less ROS following environmental stress exposure, supporting the hypothesis of a protective mechanism to mediate radical production when subject to harsh conditions. Studies concerning other physical and chemical stressors, as well as examining radical production in tardigrades upon inducing tun formation, are ongoing. Further experiments also include determining specific activities of proteinaceous antioxidants like SOD before and after these exposures.Support or Funding InformationJ.R.C was supported the SURE Program funded through the West Virginia Research Challenge Fund, and administered by the West Virginia HEPC, DSR, Award Number DSR.17.09,the WV NASA Space Grant Consortium's NASA WV Space Grant Undergraduate Fellowship Program and from a Marshall University Creative Discover & Undergraduate Research Award.D.R.J.K. and A.L.S. were supported by the National Science Foundation under Cooperative Agreement No. OIA‐1458952.

Ultrastructural analysis of the dehydrated tardigrade Hypsibius exemplaris unveils an anhydrobiotic-specific architecture

Tardigrades can cope with adverse environmental conditions by turning into anhydrobiotes with a characteristic tun shape. Tun formation is an essential morphological adaptation for tardigrade entry into the anhydrobiotic state. The tun cell structure and ultrastructure have rarely been explored in tardigrades in general and never in Hypsibius exemplaris. We used transmission electron microscopy to compare cellular organization and ultrastructures between hydrated and anhydrobiotic H. exemplaris. Despite a globally similar cell organelle structure and a number of cells not significantly different between hydrated and desiccated tardigrades, reductions in the sizes of both cells and mitochondria were detected in dehydrated animals. Moreover, in anhydrobiotes, secretory active cells with a dense endoplasmic reticulum network were observed. Interestingly, these anhydrobiote-specific cells are in a close relationship with a specific extracellular structure surrounding each cell. It is possible that this rampart-like extracellular structure resulted from the accumulation of anhydrobiotic-specific material to protect the cells. Interestingly, after five hours of rehydration, the number of secretory cells decreased, and the specific extracellular structure began to disappear. Twenty-four hours after the beginning of rehydration, the cellular structure and ultrastructure were comparable to those observed in hydrated tardigrades.

The Unique Antimicrobial Recognition and Signaling Pathways in Tardigrades with a Comparison Across Ecdysozoa

Tardigrades are microscopic animals known to withstand unfavorable abiotic conditions. These animals are also constantly exposed to biotic stresses, including parasites and internal microbiomes. However, the tardigrade immune mechanisms against these biotic stresses are largely uncharacterized. Due to the contentious phylogenetic position of tardigrades, it is not intuitive whether they possess an immune system more similar to that of arthropods (e.g., Toll, Imd, and JNK pathways of the Drosophila melanogaster antimicrobial response) or to that of nematodes (e.g., the Tir-1/Nsy-1/Sek-1/Pmk-1/Atf-7 signaling cassette [called Tir-1 pathway here]) in Caenorhabditis elegans). In this study, comparative genomic analyses were conducted to mine homologs of canonical D. melanogaster and C. elegans immune pathway genes from eight tardigrades (Echiniscoides cf. sigismundi, Echiniscus testudo, Hypsibius exemplaris, Mesobiotus philippinicus, Milnesium tardigradum, Paramacrobiotus richtersi, Richtersius cf. coronifer, and Ramazzottius varieornatus) and four non-arthropod ecdysozoans (two onychophorans: Epiperipatus sp. and Opisthopatus kwazululandi; one nematomorph: Paragordius varius; and one priapulan: Priapulus caudatus) in order to provide insights into the tardigrade antimicrobial system. No homologs of the intracellular components of the Toll pathway were detected in any of the tardigrades examined. Likewise, no homologs of most of the Imd pathway genes were detected in any of the tardigrades or any of the other non-arthropod ecdysozoans. Both the JNK and Tir-1 pathways, on the other hand, were found to be conserved across ecdysozoans. Interestingly, tardigrades had no detectable homologs of NF-κB, the major activator of antimicrobial response gene expression. Instead, tardigrades appear to possess NF-κB distantly related NFAT homologs. Overall, our results show that tardigrades have a unique gene pathway repertoire that differs from that of other ecdysozoans. Our study also provides a framework for future studies on tardigrade immune responses.

Dietary preferences and diet effects on life-history traits of tardigrades

AbstractTardigrades may be divided into the following feeding groups: herbivores, carnivores and omnivores. However, little is known about their specific feeding preferences. Here, we used a number of laboratory experiments with 18 potential food sources, representing a wide variety of organisms, to test feeding preferences, survival and fecundity of three tardigrade species, representing different feeding modes. We also tested for differences in preferences between juveniles and adults, and differences in survival between two age groups: one that started the experiment as juveniles and the other as adults. In our experiments, Milnesium inceptum is confirmed to be a carnivore, being able to reproduce only on animal prey. We also show that Hypsibius exemplaris is a herbivore, feeding on cyanobacteria, algae and fungi. Paramacrobiotus fairbanksi, on the other hand, is demonstrated to be an omnivore, feeding on cyanobacteria, algae, fungi and animals. In some cases, juveniles preferred different types of food than adults. Reproduction was strongly affected by food type. Finally, we demonstrate that tardigrades may ingest food types that they are not able to digest. Thus, gut content analysis may be misleading as a method of studying tardigrade feeding habits.

Comparison of the transcriptomes of two tardigrades with different hatching coordination

BackgroundTardigrades are microscopic organisms, famous for their tolerance against extreme environments. The establishment of rearing systems of multiple species has allowed for comparison of tardigrade physiology, in particular in embryogenesis. Interestingly, in-lab cultures of limnic species showed smaller variation in hatching timing than terrestrial species, suggesting a hatching regulation mechanism acquired by adaptation to their habitat.ResultsTo this end, we screened for coordinated gene expression during the development of two species of tardigrades, Hypsibius exemplaris and Ramazzottius varieornatus, and observed induction of the arthropod molting pathway. Exposure of ecdysteroids and juvenile hormone analog affected egg hatching but not embryonic development in only the limnic H. exemplaris.ConclusionThese observations suggest a hatching regulation mechanism by the molting pathway in H. exemplaris.

AMPK activity is required for the induction of anhydrobiosis in a tardigrade Hypsibius exemplaris, and its potential up-regulator is PP2A.

The anhydrobiotic tardigrade, Hypsibius exemplaris, was previously considered to require de novo gene expression and protein phosphatase 1 (PP1) and protein phosphatase 2A (PP2A) activity for successful anhydrobiosis. These indicate that H.exemplaris has signal transduction systems responding to desiccation stress, with the involvement of phosphorylation events. To this end, we carried out time-series phosphoproteomics of H.exemplaris exposed to mild desiccation stress and detected 48 phosphoproteins with significant differential regulations. Among them, immediate and successive reduction of phosphorylation levels of AMP-activated protein kinase (AMPK) was observed. The subsequent chemical genetic approach showed that AMPK was activated during the preconditioning stage for anhydrobiosis, and inhibition of its activity impaired successful anhydrobiosis. As PP2A is known to dephosphorylate AMPK in other organisms, we suggested that decreased phosphorylation levels of AMPK upon mild desiccation stress were caused by dephosphorylation by PP2A. Accordingly, phosphoproteomics of animals pre-treated with the PP1/PP2A inhibitor cantharidic acid (CA) lacked the decrease in phosphorylation levels of AMPK. These observations suggest that AMPK activity is required for successful anhydrobiosis in H.exemplaris, and its phosphorylation state is possibly regulated by PP2A.

Differential mechanisms of tolerance to extreme environmental conditions in tardigrades

Tardigrades, also known as water bears, are small aquatic animals that inhabit marine, fresh water or limno-terrestrial environments. While all tardigrades require surrounding water to grow and reproduce, species living in limno-terrestrial environments (e.g. Ramazzottius varieornatus) are able to undergo almost complete dehydration by entering an arrested state known as anhydrobiosis, which allows them to tolerate ionic radiation, extreme temperatures and intense pressure. Previous studies based on comparison of the genomes of R. varieornatus and Hypsibius dujardini - a less tolerant tardigrade - have pointed to potential mechanisms that may partially contribute to their remarkable ability to resist extreme physical conditions. In this work, we have further annotated the genomes of both tardigrades using a guided approach in search for novel mechanisms underlying the extremotolerance of R. varieornatus. We have found specific amplifications of several genes, including MRE11 and XPC, and numerous missense variants exclusive of R. varieornatus in CHEK1, POLK, UNG and TERT, all of them involved in important pathways for DNA repair and telomere maintenance. Taken collectively, these results point to genomic features that may contribute to the enhanced ability to resist extreme environmental conditions shown by R. varieornatus.

Acoustomicrofluidic separation of tardigrades from raw cultures for sample preparation

Abstract Tardigrades are microscopic animals widely known for their ability to survive in extreme conditions. They are the focus of current research in the fields of taxonomy, biogeography, genomics, proteomics, development, space biology, evolution and ecology. Tardigrades, such as Hypsibius exemplaris, are being advocated as a next-generation model organism for genomic and developmental studies. The raw culture of H. exemplaris usually contains tardigrades themselves, their eggs, faeces and algal food. Experimentation with tardigrades often requires the demanding and laborious separation of tardigrades from raw samples to prepare pure and contamination-free tardigrade samples. In this paper, we propose a two-step acoustomicrofluidic separation method to isolate tardigrades from raw samples. In the first step, a passive microfluidic filter composed of an array of traps is used to remove large algal clusters in the raw sample. In the second step, a surface acoustic wave-based active microfluidic separation device is used to deflect tardigrades continuously from their original streamlines inside the microchannel and thus isolate them selectively from algae and eggs. The experimental results demonstrated the efficient separation of tardigrades, with a recovery rate of 96% and an impurity of 4% algae on average in a continuous, contactless, automated, rapid and biocompatible manner.

Cellular morphology of leg musculature in the water bear Hypsibius exemplaris (Tardigrada) unravels serial homologies.

Tardigrades (water bears) are microscopic, segmented ecdysozoans with four pairs of legs. Lobopodous limbs that are similar to those seen in tardigrades are hypothesized to represent the ancestral state of Panarthropoda (Tardigrada + Onychophora + Arthropoda), and their evolutionary history is important to our understanding of ecdysozoan evolution. Equally important is our understanding of the functional morphology of these legs, which requires knowledge of their musculature. Tardigrade musculature is well documented but open questions remain. For example, while the muscular organization of each trunk segment and its legs is unique, three of the four trunk segments are nevertheless relatively homonomous. To what extent, then, do leg muscles show segmental patterns? Specifically, which leg muscles are serially repeated and which are unique? The present study addresses these questions using a combination of techniques intended to visualize both the overall layout and fine structure of leg muscles in the eutardigrade Hypsibius exemplaris. In doing so, we propose serial homologies for all leg muscles in each of the four legs and reveal new details of their cellular structure and attachment sites. We compare our results to those of previous studies and address the functional implications of specialized muscle cell morphologies.

The tardigrade damage suppressor protein binds to nucleosomes and protects DNA from hydroxyl radicals.

Tardigrades, also known as water bears, are animals that can survive extreme conditions. The tardigrade Ramazzottius varieornatus contains a unique nuclear protein termed Dsup, for damage suppressor, which can increase the resistance of human cells to DNA damage under conditions, such as ionizing radiation or hydrogen peroxide treatment, that generate hydroxyl radicals. Here we find that R. varieornatus Dsup is a nucleosome-binding protein that protects chromatin from hydroxyl radicals. Moreover, a Dsup ortholog from the tardigrade Hypsibius exemplaris similarly binds to nucleosomes and protects DNA from hydroxyl radicals. Strikingly, a conserved region in Dsup proteins exhibits sequence similarity to the nucleosome-binding domain of vertebrate HMGN proteins and is functionally important for nucleosome binding and hydroxyl radical protection. These findings suggest that Dsup promotes the survival of tardigrades under diverse conditions by a direct mechanism that involves binding to nucleosomes and protecting chromosomal DNA from hydroxyl radicals.

Detoxification of toxic heavy metals by serine protease inhibitor isolated from polluted soil

Various anthropogenic activities attribute to the addition and accumulation of heavy metals in soil which creates an environment unsuitable for all organisms. Despite such conditions, some microbial populations are able to survive and grow due to functioning of certain genes. Investigating the role of eukaryotic microbes in metal contaminated soil was the focus of this study. Culture independent approach was employed and size fractionated eukaryotic cDNA libraries (library A < 0.5 kb, library B 0.5–1.0 kb, and library C 1.0–4.0 kb) of soil were constructed from soil RNA and were screened for cadmium (Cd) tolerance by functional complementation assay by using Cd sensitive yeast mutant ycf1Δ. Screening of the library C yielded clones capable of growing in presence of Cd amended media. In the present study, one of the clones, PLCg49 was selected because of its ability to grow at high concentrations of Cd (40–100 μM). PLCg49 was also found to be highly inducible in presence of Cd where the highest fold expression was observed at 60 μM of Cd. Sequence analysis of PLCg49 showed homology with Serine Protease Inhibitor (Serpin) of the eukaryote Hypsibius dujardini. Serpins are a broad family of protease inhibitors which are majorly stress-inducible genes usually triggered by biotic and abiotic stresses and help in maintaining cell viability. Copper-sensitive (cup1Δ), Zn-sensitive (zrc1Δ) and Cobalt-sensitive (cot1Δ) yeast mutants rescued their sensitivity when transformed with PLCg49, indicating its tolerance to different heavy metals. The protease sensitive yeast mutant (pbi2Δ) transformed with PLCg49 protected from the cell lysis when grown in presence of trypsin and proteinase K confirming the role of PLCg49 as an effective serpin. Present study results suggested that PLCg49 serves as a potential metal tolerant gene, helping us to understand new pathways participating in metal tolerance.

A sustainable culture of tardigrades (Hypsibius dujardini) for astrobiological laboratory activities

Tardigrades are microscopic organisms that have gained importance in astrobiology in recent years. They have extreme survival ability and can enter into a state of latency for several years called cryptobiosis. Due to our interest in conducting experiments with tardigrades to complement the laboratory exercises with students in the course of astrobiology, we bought samples of tardigrades in the cryptobiotic state. A sample of these were cultured at room temperature (18oC) with an oxygenation system, and fed with Spirulina algae every week. We found that in a few weeks tardigrades were producing eggs normally. This species, H. dujardini, has been cultured before and it is a good sample for culture as we saw. This species provides a good sustainable culture and is inexpensive and easy to produce tardigrades for didactic purposes.

Carbonylation accumulation of the Hypsibius exemplaris anhydrobiote reveals age-associated marks

Together with nematodes and rotifers, tardigrade belong to micrometazoans that can cope with environmental extremes such as UV and solar radiations, dehydration, supercooling or overheating. Tardigrade can resist the harshest conditions by turning to cryptobiosis, an anhydrobiotic state that results from almost complete dehydration and is characterized by an ametabolic status. Although reports have challenged the molecular basis of the mechanisms underlying genomic injury resistance, little is yet known regarding the possible involvement of other tardigrade macromolecules in injury during a stress experience. In this report, we show that the tardigrade Hypsibius exemplaris can accumulate molecular damages by means of in situ detection of carbonyls. Furthermore, we demonstrate that living tardigrade can accumulate carbonylation. Finally, we reveal that anhydrobiotic tardigrade can be constitutively affected by carbonylation that marks aging in other metazoans.

The Emergence of the Tardigrade Hypsibius exemplaris as a Model System.

The success of scientists in revealing biological mechanisms has depended in large part on choosing tractable model systems. In 1997, molecular phylogenetics revealed that two of biology's most tractable models-Caenorhabditis elegans and Drosophila-are much more closely related to each other than had been thought previously. I began to explore whether any of the little-studied members of this branch of the tree of life might serve as a new model for comparative biology that could make use of the rich and ongoing sources of information flowing from C. elegans and Drosophila research. Tardigrades, also known as water bears, make up a phylum of microscopic animals. The tardigrade Hypsibius exemplaris (recently disambiguated from a closely related species, Hypsibius dujardini) can be maintained in laboratories and has a generation time of <2 wk at room temperature. Stocks of animals can be stored frozen and revived. The animals and their embryos are optically clear, and embryos are laid in groups, with each synchronous clutch of embryos laid in a clear molt. We have developed techniques for laboratory study of this system, including methods for microinjection of animals, immunolocalization, in situ hybridization, RNA interference, transcriptomics, and methods for identifying proteins that mediate tolerance to extreme environments. Here, I review the development of this animal as an emerging model system, as well as recent molecular studies aimed at understanding the evolution of developmental mechanisms that underpin the evolution of animal form and at understanding how biological materials can survive extreme environments.

Desiccation of Hypsibius exemplaris.

Many species of tardigrades can survive severe water loss, but different species tolerate different desiccation conditions. Hypsibius exemplaris is able to survive desiccation after an initial period of slow drying, as described here. This protocol will likely work for other tardigrade species as well. Drying of tardigrades can be used for probing the mechanistic underpinnings of desiccation tolerance, as well as for practical purposes such as shipping and long-term storage of the animals.

Microinjection of dsRNA in Tardigrades.

Classical genetic analysis in the tardigrade Hypsibius exemplaris is a challenge because these animals are parthenogens. The publication of the H. exemplaris genome has facilitated the study of targeted genes by RNA interference (RNAi), a robust mechanism to disrupt gene function. This protocol describes microinjection of double-stranded RNA (dsRNA) in tardigrades using techniques adapted from protocols originally developed in Caenorhabditis elegans. A DNA template (either genomic or cDNA) is used to prepare dsRNA, to which T7 polymerase binding sites are added at the 5' end of each strand. The dsRNA is injected into adult tardigrades, preferably targeting the gonad or intestine. Injected adults are allowed to recover in spring water and then transferred to culture dishes or individual wells of a 96-well plate.

Laboratory Culture of Hypsibius exemplaris

I have reared a culture of the tardigrade Hypsibius exemplaris for 30 years, since 1987. Here, I present my culture protocol.

Live Imaging of Tardigrade Embryonic Development by Differential Interference Contrast Microscopy.

The tardigrade Hypsibius exemplaris was chosen as a model system in part because embryos and animals are optically clear at all stages, facilitating the viewing and filming of internal processes. Multiplane video recordings under differential interference contrast (DIC) microscopy have allowed early embryonic cell lineages to be reconstructed through seven rounds of division and have revealed invariant patterns of asymmetric cell divisions, nuclear migrations, and cell migrations. Here, we present a protocol for filming embryonic development of H. exemplaris by DIC microscopy.

Fluorescent Cell Staining Methods for Living Hypsibius exemplaris Embryos.

The tardigrade Hypsibius exemplaris was chosen as a model system in part because animals and embryos are optically clear at all stages, facilitating the visualization of events in living material. Here we report new methods for introducing fluorescent dyes into developing H. exemplaris embryos, including methods for fluorescently marking mitochondria, lysosomes, membranes, and nuclei. The development of these techniques suggests approaches for attempting to introduce other molecules into embryos.

Embryonic Immunostaining for the Tardigrade Hypsibius exemplaris

Immunostaining is a method used to visualize the localization of proteins in fixed tissue. Many antibodies are available that recognize specific proteins in a wide diversity of organisms, which makes this method ideal for investigating gene expression patterns in nonmodel animal systems. This protocol describes immunostaining for studies of embryogenesis in the tardigrade Hypsibius exemplaris.