Comparative transcriptomics reveal a novel tardigrade-specific DNA-binding protein induced in response to ionizing radiation.

Tardigrades are microscopic animals renowned for their ability to withstand extreme conditions, including high doses of ionizing radiation (IR). To better understand their radio-resistance, we first characterized induction and repair of DNA double- and single-strand breaks after exposure to IR in the model species Hypsibius exemplaris. Importantly, we found that the rate of single-strand breaks induced was roughly equivalent to that in human cells, suggesting that DNA repair plays a predominant role in tardigrades' radio-resistance. To identify novel tardigrade-specific genes involved, we next conducted a comparative transcriptomics analysis across three different species. In all three species, many DNA repair genes were among the most strongly overexpressed genes alongside a novel tardigrade-specific gene, which we named Tardigrade DNA damage Response 1 (TDR1). We found that TDR1 protein interacts with DNA and forms aggregates at high concentration suggesting it may condensate DNA and preserve chromosome organization until DNA repair is accomplished. Remarkably, when expressed in human cells, TDR1 improved resistance to Bleomycin, a radiomimetic drug. Based on these findings, we propose that TDR1 is a novel tardigrade-specific gene conferring resistance to IR. Our study sheds light on mechanisms of DNA repair helping cope with high levels of DNA damage inflicted by IR.

Surviving extreme radiation.

Tiny animals known as tardigrades use a combination of DNA repair machinery and a novel protein to mend their genome after intense ionizing radiation.

The organisation of the digestive, excretory and reproductive systems in cysts of the freshwater tardigrade Thulinius ruffoi (Parachela, Isohypsibioidea: Doryphoribiidae).

Tardigrades are invertebrates known to science for over 250 years. Although the ability of some species of tardigrades to form cysts has been reported, little is known about the encystment and internal organisation of the cysts. During cyst formation, contraction of the body affects the internal organs' morphology. The organs are compressed and have a compact appearance. The organisation of the digestive system, associated structures, and the reproductive system are analysed in cysts on indefinite and well-defined encystment periods - up to eleven months. The digestive system of encysted animals was organised into three main parts - a foregut, a midgut, and a hindgut. The presence of digestive system-associated structures, such as buccal glands or muscles, was noted and described. The excretory organs, called Malpighian tubules, open into the zone between the midgut and the hindgut. Furthermore, the oviduct opens into the hindgut. The first analysis of the reproductive system of cysts at the ultrastructural level is presented here, revealing interesting and undescribed aspects related to the physiology. Besides the anatomical and histological examination, the morphology and changes that occur during cyst formation are described.

The tardigrade Hypsibius exemplaris dramatically upregulates DNA repair pathway genes in response to ionizing radiation.

Tardigrades can survive remarkable doses of ionizing radiation, up to about 1,000 times the lethal dose for humans. How they do so is incompletely understood. We found that the tardigrade Hypsibius exemplaris suffers DNA damage upon gamma irradiation, but the damage is repaired. We show that this species has a specific and robust response to ionizing radiation: irradiation induces a rapid upregulation of many DNA repair genes. This upregulation is unexpectedly extreme-making some DNA repair transcripts among the most abundant transcripts in the animal. By expressing tardigrade genes in bacteria, we validate that increased expression of some repair genes can suffice to increase radiation tolerance. We show that at least one such gene is important in vivo for tardigrade radiation tolerance. We hypothesize that the tardigrades' ability to sense ionizing radiation and massively upregulate specific DNA repair pathway genes may represent an evolved solution for maintaining DNA integrity.

Chemobiosis reveals tardigrade tun formation is dependent on reversible cysteine oxidation.

Tardigrades, commonly known as 'waterbears', are eight-legged microscopic invertebrates renowned for their ability to withstand extreme stressors, including high osmotic pressure, freezing temperatures, and complete desiccation. Limb retraction and substantial decreases to their internal water stores results in the tun state, greatly increasing their ability to survive. Emergence from the tun state and/or activity regain follows stress removal, where resumption of life cycle occurs as if stasis never occurred. However, the mechanism(s) through which tardigrades initiate tun formation is yet to be uncovered. Herein, we use chemobiosis to demonstrate that tardigrade tun formation is mediated by reactive oxygen species (ROS). We further reveal that tuns are dependent on reversible cysteine oxidation, and that this reversible cysteine oxidation is facilitated by the release of intracellular reactive oxygen species (ROS). We provide the first empirical evidence of chemobiosis and map the initiation and survival of tardigrades via osmobiosis, chemobiosis, and cryobiosis. In vivo electron paramagnetic spectrometry suggests an intracellular release of reactive oxygen species following stress induction; when this release is quenched through the application of exogenous antioxidants, the tardigrades can no longer survive osmotic stress. Together, this work suggests a conserved dependence of reversible cysteine oxidation across distinct tardigrade cryptobioses.

Tardigrades in the marine plastisphere: New hitchhikers surfing plastics.

Tardigrades are remarkable microorganisms known for their extraordinary resilience in diverse environments, including extreme conditions such as outer space. They are known for their interactions with natural substrates in terrestrial and aquatic systems, but have remained largely unexplored in relation to marine plastics. This study aims to investigate the colonization of plastics, ranging from fossil fuel-based to bioplastics, in the coastal zones of four countries (Brazil, Ireland, France and Italy). Here, we report the first documented occurrence of tardigrades colonizing plastic substrates. We identified five amplicon sequence variants (ASVs) belonging to the Tardigrada phylum, specifically in a post-consumer polypropylene, in the coastal zone of Galway, Ireland. This discovery raises questions about the characteristics of different plastics influencing on tardigrades' adhesion. Tardigrades hitchhiking on plastics in the oceans could expand their habitat range, possibly displacing native species and altering trophic interactions, with potential consequences for the overall biodiversity.

Organisation of the nervous system in cysts of the freshwater tardigrade Thulinius ruffoi (Parachela, Isohypsibioidea: Doryphoribiidae).

Encystment is a natural process that involves cyst formation, and at least some species of tardigrades can form cysts. However, the encystment process and cyst structure among tardigrades are still poorly understood. Despite some aspects of the encysted animals' systems organisation being examined in the past, the morphology and structure of the nervous system have never been thoroughly investigated. This study covers anatomical, histological and morphological details and proposes physiological aspects of the nervous system in encysted Thulinius ruffoi up to 11 months duration in encystment. This is the first record of the nervous system organisation in a species belonging to the family Doryphoribiidae. The cyst formation results in morphological changes in the nervous system. It comprises central and peripheral elements, which may be observable even after many months since the cyst formation. Based on the nervous system's organisation in cysts, there is no sign that histolysis is a part of encystment.

Experimental investigation of the creep damage evolution of coal rock around gas extraction boreholes at different water contents.

The creep process of the coal rock around the extraction boreholes under stress-water coupling is an important factor affecting the stability of the boreholes. To study the influence of the water content of perimeter of the coal rock around the boreholes on its creep damage, a creep intrinsic model considering water damage was established by introducing the plastic element model from the Nishihara model. To study the steady-state strain and damage evolution of coal rocks containing pores, and verify the practicality of the model, a graded loading water-bearing creep test was designed to explore the role of different water-bearing conditions in the creep process. The following conclusions were obtained: 1) water has a physical erosion and softening water wedge effect on the perimeter of the coal rock around the boreholes, which affects the loading axial strain and displacement of the perforated specimens; 2) an increase in water content reduces the time taken for perforated specimens to enter the creep phase, making the accelerated creep phase come earlier; 3) the parameters of the water damage model are considered to be exponentially related with the water content. The experimental data are similar to the results of the model parameters, and the model shows some practicality; 4) the damage variables in the accelerated creep phase increase rapidly throughout the creep process, leading to local instability in the borehole. The findings of the study provide important theoretical implications for the study of instability in gas extraction boreholes.

Time-series transcriptomic screening of factors contributing to the cross-tolerance to UV radiation and anhydrobiosis in tardigrades.

BACKGROUND: Tardigrades are microscopic animals that are capable of tolerating extreme environments by entering a desiccated state of suspended animation known as anhydrobiosis. While antioxidative stress proteins, antiapoptotic pathways and tardigrade-specific intrinsically disordered proteins have been implicated in the anhydrobiotic machinery, conservation of these mechanisms is not universal within the phylum Tardigrada, suggesting the existence of overlooked components. RESULTS: Here, we show that a novel Mn-dependent peroxidase is an important factor in tardigrade anhydrobiosis. Through time-series transcriptome analysis of Ramazzottius varieornatus specimens exposed to ultraviolet light and comparison with anhydrobiosis entry, we first identified several novel gene families without similarity to existing sequences that are induced rapidly after stress exposure. Among these, a single gene family with multiple orthologs that is highly conserved within the phylum Tardigrada and enhances oxidative stress tolerance when expressed in human cells was identified. Crystallographic study of this protein suggested Zn or Mn binding at the active site, and we further confirmed that this protein has Mn-dependent peroxidase activity in vitro. CONCLUSIONS: Our results demonstrated novel mechanisms for coping with oxidative stress that may be a fundamental mechanism of anhydrobiosis in tardigrades. Furthermore, localization of these sets of proteins mainly in the Golgi apparatus suggests an indispensable role of the Golgi stress response in desiccation tolerance.

Desiccation-tolerance and globular proteins adsorb similar amounts of water.

When exposed to desiccation stress, extremotolerant organisms from all domains of life produce protective disordered proteins with the potential to inform the design of excipients for formulating biologics and industrial enzymes. However, the mechanism(s) of desiccation protection remain largely unknown. To investigate the role of water sorption in desiccation protection, we use thermogravimetric analysis to study water adsorption by two desiccation-tolerance proteins, cytosolic abundant heat soluble protein D from tardigrades and late embryogenesis abundant protein 4 from the anhydrobiotic midge Polypedilum vanderplanki, and, as a control, the globular B1 domain of staphylococcal protein G. All samples adsorb similar amounts of water, suggesting that modulated water retention is not responsible for dehydration protection by desiccation-tolerance proteins.

Naturally occurring fluorescence protects the eutardigrade Paramacrobiotus sp. from ultraviolet radiation.

Naturally occurring fluorescence has been observed in multiple species ranging from bacteria to birds. In macroscopic animals such as birds, fluorescence provides a visual communication signal. However, the functional significance of this phenomenon is unknown in most cases. Though photoprotection is attributed to fluorescence under ultraviolet (UV) light in some organisms, it lacks direct experimental evidence. Here, we demonstrate naturally occurring fluorescence under UV light in a eutardigrade belonging to the genus Paramacrobiotus. Using a natural variant that lacks fluorescence, we show that the fluorescence confers tolerance to lethal UV radiation. Remarkably, the fluorescent extract from Paramacrobiotus sp. could protect the UV-sensitive tardigrade Hypsibius exemplaris and nematode Caenorhabditis elegans from germicidal UV radiation. We propose that Paramacrobiotus sp. possess a protective fluorescent shield that absorbs harmful UV radiation and emits harmless blue light.

Expression of a Tardigrade Dsup Gene Enhances Genome Protection in Plants.

DNA damage is one of the most impactful events in living organisms, leading to DNA sequence changes (mutation) and disruption of biological processes. A study has identified a protein called Damage Suppressor Protein (Dsup) in the tardigrade Ramazzotius varieornatus that has shown to reduce the effects of radiation damage in human cell cultures (Hashimoto in Nature Communications 7:12808, 2016). We have generated tobacco plants that express the codon-optimized tardigrade Dsup gene and examined their responses when treated with mutagenic chemicals, ultraviolet (UV) and ionizing radiations. Our studies showed that compared to the control plants, the Dsup-expressing plants grew better in the medium containing mutagenic ethylmethane sulfonate (EMS). RT-qPCR detected distinct expression patterns of endogenous genes involved in DNA damage response and repair in the Dsup plants in response to EMS, bleomycin, UV-C and X-ray radiations. Comet assays revealed that the nuclei from the Dsup plants appeared more protected from UV and X-ray damages than the control plants. Overall, our studies demonstrated that Dsup gene expression enhanced tolerance of plants to genomutagenic stress. We suggest the feasibility of exploring genetic resources from extremotolerant species such as tardigrades to impart plants with tolerance to stressful environments for future climate changes and human space endeavors.

An integrative redescription of Hypsibius dujardini (Doyère, 1840), the nominal taxon for Hypsibioidea (Tardigrada: Eutardigrada).

A laboratory strain identified as "Hypsibius dujardini" is one of the best studied tardigrade strains: it is widely used as a model organism in a variety of research projects, ranging from developmental and evolutionary biology through physiology and anatomy to astrobiology. Hypsibius dujardini, originally described from the Île-de-France by Doyère in the first half of the 19th century, is now the nominal species for the superfamily Hypsibioidea. The species was traditionally considered cosmopolitan despite the fact that insufficient, old and sometimes contradictory descriptions and records prevented adequate delineations of similar Hypsibius species. As a consequence, H. dujardini appeared to occur globally, from Norway to Samoa. In this paper, we provide the first integrated taxonomic redescription of H. dujardini. In addition to classic imaging by light microscopy and a comprehensive morphometric dataset, we present scanning electron photomicrographs, and DNA sequences for three nuclear markers (18S rRNA, 28S rRNA, ITS-2) and one mitochondrial marker (COI) that are characterised by various mutation rates. The results of our study reveal that a commercially available strain that is maintained in many laboratories throughout the world, and assumed to represent H. dujardini sensu stricto, represents, in fact, a new species: H. exemplaris sp. nov. Redescribing the nominal taxon for Hypsibiidae, we also redefine the family and amend the definitions of the subfamily Hypsibiinae and the genus Hypsibius. Moreover, we transfer H. arcticus (Murray, 1907) and Hypsibius conifer Mihelcic, 1938 to the genus Ramazzottius since the species exhibit claws and eggs of the Ramazzottius type. Finally, we designate H. fuhrmanni as subjectively invalid because the extremely poor description precludes identifying neotype material.

Integrative taxonomy identifies Macrobiotus papei, a new tardigrade species of the Macrobiotus hufelandi complex (Eutardigrada: Macrobiotidae) from the Udzungwa Mountains National Park (Tanzania).

In this paper we describe Macrobiotus papei, a new species of the Macrobiotus hufelandi complex from the Udzungwa Mountains National Park in Tanzania. Our research included the traditional morphology-based taxonomic analysis, supported by morphometrics, light and scanning electron microscopy imaging, as well as analysis of nucleotide sequences of three nuclear (18S rRNA, 28S rRNA, ITS-2) and one mitochondrial (COI) marker. The molecular sequences provided a more accurate description and independent verification of the taxonomic status of M. papei sp. nov. Such integrative approach requires a considerable number of individuals and eggs, which we obtained by establishing a laboratory culture of the new species. Our analyses revealed that M. papei sp. nov., by having the flexible filaments on the terminal discs of the egg processes, is most similar to Macrobiotus paulinae Stec et al., 2015 from Africa and Macrobiotus polypiformis Roszkowska et al., 2017 from South America. However, the new species can be easily distinguished from both these species by patches of cuticular granulation on the external and internal surface of legs I-III (only external patches present in in M. paulinae and M. polypiformis), two separate lateral transversal ridges with a rounded median tooth between instead of a single thin ventral transverse ridge in the third band of teeth in the oral cavity, smooth terminal discs and filaments on the egg processes (terminal discs and filaments covered by aggregations of small microgranules in M. paulinae and M. polypiformis), and by some morphometric characters of both adults and eggs.

An integrative description of Macrobiotus shonaicus sp. nov. (Tardigrada: Macrobiotidae) from Japan with notes on its phylogenetic position within the hufelandi group.

Tardigrade research in Japan dates back over 100 years, and to date, 167 species of this ecdysozoan phylum have been reported from the country. Of these species, the Macrobiotus hufelandi complex has been represented only by the nominal taxon of this group, Macrobiotus hufelandi. In this article, a new species of the hufelandi group from Japan, Macrobiotus shonaicus sp. nov., is described using integrative taxonomy. In addition to the detailed morphological and morphometric data, obtained using phase contrast light microscopy (PCM) and scanning electron microscopy (SEM), we provide DNA sequences of four molecular markers (both nuclear and mitochondrial). The new species belongs to the persimilis subgroup and is most similar to M. anemone from USA, M. naskreckii from Mozambique, and M. patagonicus from Argentina, but it can be easily distinguished from these species by the presence of thin flexible filaments on terminal discs of the egg process. By the latter character, the new species is most similar to M. paulinae and M. polypiformis, but it can be easily distinguished from them by having a solid egg surface between egg processes (i.e., without pores or reticulum). A phylogenetic analysis of available DNA sequences of the COI marker for the hufelandi group revealed that the new species clusters with the two other species that exhibit filaments on egg process discs (M. paulinae and M. polypiformis) and with two species that have entire egg processes modified into filaments (M. kristenseni and M. scoticus). All five species form a clade distinct from all other sequenced species of the hufelandi group with typical mushroom- or inverted goblet-shaped egg processes, which may suggest that the ancestor of the five species with atypical egg processes had a mutation allowing derivations from the mushroom or inverted chalice-like shape of egg processes.

Exceptional in vivo catabolism of neurodegeneration-related aggregates.

Neurodegenerative diseases are linked to a systemic enzyme resistance of toxic aggregated molecules and their pathological consequences. This paper presents a unique phenomenon that Philodina acuticornis, a bdelloid rotifer, is able to catabolize different types of neurotoxic peptide and protein aggregates (such as beta-amyloids /Aß/, alpha-synuclein, and prion) without suffering any damage. P. acuticornis is capable of using these aggregates as an exclusive energy source (i.e., as 'food', identified in the digestive system and body) in a hermetically isolated microdrop environment, increasing their survival. As regards Aß1-42, five other bdelloid rotifer species were also found to be able to perform this phenomenon. Based on our experiments, the Aß1-42-treated bdelloid rotifers demonstrate significantly increased survival (e.g. mean lifespan = 51 ± 2.71 days) compared to their untreated controls (e.g. mean lifespan = 14 ± 2.29 days), with similar improvements in a variety of phenotypic characteristics. To our knowledge, no other animal species have so far been reported to have a similar capability. For all other microscopic species tested, including monogonant rotifers and non-rotifers, the treatment with Aß1-42 aggregates proved to be either toxic or simply ineffective. This paper describes and proves the existence of an unprecedented in vivo catabolic capability of neurotoxic aggregates by bdelloid rotifers, with special focus on P. acuticornis. Our results may provide the basis for a new preclinical perspective on therapeutic research in human neurodegenerative diseases.

Cytochrome P450 diversity in the tree of life.

Sequencing in all areas of the tree of life has produced >300,000 cytochrome P450 (CYP) sequences that have been mined and collected. Nomenclature has been assigned to >41,000 CYP sequences and the majority of the remainder has been sorted by BLAST searches into clans, families and subfamilies in preparation for naming. The P450 sequence space is being systematically explored and filled in. Well-studied groups like vertebrates are covered in greater depth while new insights are being added into uncharted territories like horseshoe crab (Limulus polyphemus), tardigrades (Hypsibius dujardini), velvet worm (Euperipatoides_rowelli), and basal land plants like hornworts, liverworts and mosses. CYPs from the fungi, one of the most diverse groups, are being explored and organized as nearly 800 fungal species are now sequenced. The CYP clan structure in fungi is emerging with 805 CYP families sorting into 32 CYP clans. >3000 bacterial sequences are named, mostly from terrestrial or freshwater sources. Of 18,379 bacterial sequences downloaded from the CYPED database, all are >43% identical to named CYPs. Therefore, they fit in the 602 named P450 prokaryotic families. Diversity in this group is becoming saturated, however 25% of 3305 seawater bacterial P450s did not match known P450 families, indicating marine bacterial CYPs are not as well sampled as land/freshwater based bacterial CYPs. Future sequencing plans of the Genome 10K project, i5k and GIGA (Global Invertebrate Genomics Alliance) are expected to produce more than one million cytochrome P450 sequences by 2020. This article is part of a Special Issue entitled: Cytochrome P450 biodiversity and biotechnology, edited by Erika Plettner, Gianfranco Gilardi, Luet Wong, Vlada Urlacher, Jared Goldstone.

Tolerance to Gamma Radiation in the Marine Heterotardigrade, Echiniscoides sigismundi.

Tardigrades belong to the most radiation tolerant animals on Earth, as documented by a number of studies using both low-LET and high-LET ionizing radiation. Previous studies have focused on semi-terrestrial species, which are also very tolerant to desiccation. The predominant view on the reason for the high radiation tolerance among these semi-terrestrial species is that it relies on molecular mechanisms that evolved as adaptations for surviving dehydration. In this study we report the first study on radiation tolerance in a marine tardigrade, Echiniscoides sigismundi. Adult specimens in the hydrated active state were exposed to doses of gamma radiation from 100 to 5000 Gy. The results showed little effect of radiation at 100 and 500 Gy but a clear decline in activity at 1000 Gy and higher. The highest dose survived was 4000 Gy, at which ca. 8% of the tardigrades were active 7 days after irradiation. LD50 in the first 7 days after irradiation was in the range of 1100-1600 Gy. Compared to previous studies on radiation tolerance in semi-terrestrial and limnic tardigrades, Echiniscoides sigismundi seems to have a lower tolerance. However, the species still fits into the category of tardigrades that have high tolerance to both desiccation and radiation, supporting the hypothesis that radiation tolerance is a by-product of adaptive mechanisms to survive desiccation. More studies on radiation tolerance in tardigrade species adapted to permanently wet conditions, both marine and freshwater, are needed to obtain a more comprehensive picture of the patterns of radiation tolerance.

Tolerance to Gamma Radiation in the Tardigrade Hypsibius dujardini from Embryo to Adult Correlate Inversely with Cellular Proliferation.

Tardigrades are highly tolerant to desiccation and ionizing radiation but the mechanisms of this tolerance are not well understood. In this paper, we report studies on dose responses of adults and eggs of the tardigrade Hypsibius dujardini exposed to gamma radiation. In adults the LD50/48h for survival was estimated at ~ 4200 Gy, and doses higher than 100 Gy reduced both fertility and hatchability of laid eggs drastically. We also evaluated the effect of radiation (doses 50 Gy, 200 Gy, 500 Gy) on eggs in the early and late embryonic stage of development, and observed a reduced hatchability in the early stage, while no effect was found in the late stage of development. Survival of juveniles from irradiated eggs was highly affected by a 500 Gy dose, both in the early and the late stage. Juveniles hatched from eggs irradiated at 50 Gy and 200 Gy developed into adults and produced offspring, but their fertility was reduced compared to the controls. Finally we measured the effect of low temperature during irradiation at 4000 Gy and 4500 Gy on survival in adult tardigrades, and observed a slight delay in the expressed mortality when tardigrades were irradiated on ice. Since H. dujardini is a freshwater tardigrade with lower tolerance to desiccation compared to limno-terrestrial tardigrades, the high radiation tolerance in adults, similar to limno-terrestrial tardigrades, is unexpected and seems to challenge the idea that desiccation and radiation tolerance rely on the same molecular mechanisms. We suggest that the higher radiation tolerance in adults and late stage embryos of H. dujardini (and in other studied tardigrades) compared to early stage embryos may partly be due to limited mitotic activity, since tardigrades have a low degree of somatic cell division (eutely), and dividing cells are known to be more sensitive to radiation.