A comparative ultrastructure study of the tardigrade Ramazzottius varieornatus in the hydrated state, after desiccation and during the process of rehydration.

Tardigrades can survive hostile environments such as desiccation by adopting a state of anhydrobiosis. Numerous tardigrade species have been described thus far, and recent genome and transcriptome analyses revealed that several distinct strategies were employed to cope with harsh environments depending on the evolutionary lineages. Detailed analyses at the cellular and subcellular levels are essential to complete these data. In this work, we analyzed a tardigrade species that can withstand rapid dehydration, Ramazzottius varieornatus. Surprisingly, we noted an absence of the anhydrobiotic-specific extracellular structure previously described for the Hypsibius exemplaris species. Both Ramazzottius varieornatus and Hypsibius exemplaris belong to the same evolutionary class of Eutardigrada. Nevertheless, our observations reveal discrepancies in the anhydrobiotic structures correlated with the variation in the anhydrobiotic mechanisms.

Single-step generation of homozygous knockout/knock-in individuals in an extremotolerant parthenogenetic tardigrade using DIPA-CRISPR.

Tardigrades are small aquatic invertebrates known for their remarkable tolerance to diverse extreme stresses. To elucidate the in vivo mechanisms underlying this extraordinary resilience, methods for genetically manipulating tardigrades have long been desired. Despite our prior success in somatic cell gene editing by microinjecting Cas9 ribonucleoproteins (RNPs) into the body cavity of tardigrades, the generation of gene-edited individuals remained elusive. In this study, employing an extremotolerant parthenogenetic tardigrade species, Ramazzottius varieornatus, we established conditions that led to the generation of gene-edited tardigrade individuals. Drawing inspiration from the direct parental CRISPR (DIPA-CRISPR) technique employed in several insects, we simply injected a concentrated Cas9 RNP solution into the body cavity of parental females shortly before their initial oviposition. This approach yielded gene-edited G0 progeny. Notably, only a single allele was predominantly detected at the target locus for each G0 individual, indicative of homozygous mutations. By co-injecting single-stranded oligodeoxynucleotides (ssODNs) with Cas9 RNPs, we achieved the generation of homozygously knocked-in G0 progeny, and these edited alleles were inherited by G1/G2 progeny. This is the first example of heritable gene editing in the entire phylum of Tardigrada. This establishment of a straightforward method for generating homozygous knockout/knock-in individuals not only facilitates in vivo analyses of the molecular mechanisms underpinning extreme tolerance, but also opens up avenues for exploring various topics, including Evo-Devo, in tardigrades.

Tardigrade secretory proteins protect biological structures from desiccation.

Tardigrades, microscopic animals that survive a broad range of environmental stresses, express a unique set of proteins termed tardigrade-specific intrinsically disordered proteins (TDPs). TDPs are often expressed at high levels in tardigrades upon desiccation, and appear to mediate stress adaptation. Here, we focus on the proteins belonging to the secreted family of tardigrade proteins termed secretory-abundant heat soluble ("SAHS") proteins, and investigate their ability to protect diverse biological structures. Recombinantly expressed SAHS proteins prevent desiccated liposomes from fusion, and enhance desiccation tolerance of E. coli and Rhizobium tropici upon extracellular application. Molecular dynamics simulation and comparative structural analysis suggest a model by which SAHS proteins may undergo a structural transition upon desiccation, in which removal of water and solutes from a large internal cavity in SAHS proteins destabilizes the beta-sheet structure. These results highlight the potential application of SAHS proteins as stabilizing molecules for preservation of cells.

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.

Tardigrades: Trained to be hardy in the face of DNA damage.

Tardigrades withstand ionizing irradiation levels ∼500 times higher than humans can tolerate. Two recent papers shed light on how this might be achieved - via the transcriptional induction of DNA repair genes, the induction of a radioprotective DNA-binding protein, and possibly also the heightened capacity of repair proteins.

The tardigrade-derived mitochondrial abundant heat soluble protein improves adipose-derived stem cell survival against representative stressors.

Human adipose-derived stem cell (ASC) grafts have emerged as a powerful tool in regenerative medicine. However, ASC therapeutic potential is hindered by stressors throughout their use. Here we demonstrate the transgenic expression of the tardigrade-derived mitochondrial abundant heat soluble (MAHS) protein for improved ASC resistance to metabolic, mitochondrial, and injection shear stress. In vitro, MAHS-expressing ASCs demonstrate up to 61% increased cell survival following 72 h of incubation in phosphate buffered saline containing 20% media. Following up to 3.5% DMSO exposure for up to 72 h, a 14-49% increase in MAHS-expressing ASC survival was observed. Further, MAHS expression in ASCs is associated with up to 39% improved cell viability following injection through clinically relevant 27-, 32-, and 34-gauge needles. Our results reveal that MAHS expression in ASCs supports survival in response to a variety of common stressors associated with regenerative therapies, thereby motivating further investigation into MAHS as an agent for stem cell stress resistance. However, differentiation capacity in MAHS-expressing ASCs appears to be skewed in favor of osteogenesis over adipogenesis. Specifically, activity of the early bone formation marker alkaline phosphatase is increased by 74% in MAHS-expressing ASCs following 14 days in osteogenic media. Conversely, positive area of the neutral lipid droplet marker BODIPY is decreased by up to 10% in MAHS-transgenic ASCs following 14 days in adipogenic media. Interestingly, media supplementation with up to 40 mM glucose is sufficient to restore adipogenic differentiation within 14 days, prompting further analysis of mechanisms underlying interference between MAHS and differentiation processes.

Conserved and specific gene expression patterns in the embryonic development of tardigrades.

Tardigrades, commonly known as water bears, are enigmatic organisms characterized by their remarkable resilience to extreme environments despite their simple and compact body structure. To date, there is still much to understand about their evolutionary and developmental features contributing to their special body plan and abilities. This research provides preliminary insights on the conserved and specific gene expression patterns during embryonic development of water bears, focusing on the species Hypsibius exemplaris. The developmental dynamic expression analysis of the genes with various evolutionary age grades indicated that the mid-conserved stage of H. exemplaris corresponds to the period of ganglia and midgut development, with the late embryonic stage showing a transition from non-conserved to conserved state. Additionally, a comparison with Drosophila melanogaster highlighted the absence of certain pathway nodes in development-related pathways, such as Maml and Hairless, which are respectively the transcriptional co-activator and co-repressor of NOTCH regulated genes. We also employed Weighted Gene Co-expression Network Analysis (WGCNA) to investigate the expression patterns of tardigrade-specific genes during embryo development. Our findings indicated that the module containing the highest proportion of tardigrade-specific genes (TSGs) exhibits high expression levels before the mid-conserved stage, potentially playing a role in glutathione and lipid metabolism. These functions may be associated to the ecdysone synthesis and storage cell formation, which is unique to tardigrades.

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.

Population growth of two limno-terrestrial Antarctic microinvertebrates in different aqueous soil media.

Terrestrial microinvertebrates provide important carbon and nutrient cycling roles in soil environments, particularly in Antarctica where larger macroinvertebrates are absent. The environmental preferences and ecology of rotifers and tardigrades in terrestrial environments, including in Antarctica, are not as well understood as their temperate aquatic counterparts. Developing laboratory cultures is critical to provide adequate numbers of individuals for controlled laboratory experimentation. In this study, we explore aspects of optimising laboratory culturing for two terrestrially sourced Antarctic microinvertebrates, a rotifer (Habrotrocha sp.) and a tardigrade (Acutuncus antarcticus). We tested a soil elutriate and a balanced salt solution (BSS) to determine their suitability as culturing media. Substantial population growth of rotifers and tardigrades was observed in both media, with mean rotifer population size increasing from 5 to 448 ± 95 (soil elutriate) and 274 ± 78 (BSS) individuals over 60 days and mean tardigrade population size increasing from 5 to 187 ± 65 (soil elutriate) and 138 ± 37 (BSS) over 160 days. We also tested for optimal dilution of soil elutriate in rotifer cultures, with 20-80% dilutions producing the largest population growth with the least variation in the 40% dilution after 36 days. Culturing methods developed in this study are recommended for use with Antarctica microinvertebrates and may be suitable for similar limno-terrestrial microinvertebrates from other regions.

Elevated external temperature affects cell ultrastructure and heat shock proteins (HSPs) in Paramacrobiotus experimentalis Kaczmarek, Mioduchowska, Poprawa, & Roszkowska, 2020.

Increasing temperature influences the habitats of various organisms, including microscopic invertebrates. To gain insight into temperature-dependent changes in tardigrades, we isolated storage cells exposed to various temperatures and conducted biochemical and ultrastructural analysis in active and tun-state Paramacrobiotus experimentalis Kaczmarek, Mioduchowska, Poprawa, & Roszkowska, 2020. The abundance of heat shock proteins (HSPs) and ultrastructure of the storage cells were examined at different temperatures (20 °C, 30 °C, 35 °C, 37 °C, 40 °C, and 42 °C) in storage cells isolated from active specimens of Pam. experimentalis. In the active animals, upon increase in external temperature, we observed an increase in the levels of HSPs (HSP27, HSP60, and HSP70). Furthermore, the number of ultrastructural changes in storage cells increased with increasing temperature. Cellular organelles, such as mitochondria and the rough endoplasmic reticulum, gradually degenerated. At 42 °C, cell death occurred by necrosis. Apart from the higher electron density of the karyoplasm and the accumulation of electron-dense material in some mitochondria (at 42 °C), almost no changes were observed in the ultrastructure of tun storage cells exposed to different temperatures. We concluded that desiccated (tun-state) are resistant to high temperatures, but not active tardigrades (survival rates of tuns after 24 h of rehydration: 93.3% at 20 °C, 60.0% at 35 °C, 33.3% at 37 °C, 33.3% at 40 °C, and 20.0% at 42 °C).

Labile assembly of a tardigrade protein induces biostasis.

Tardigrades are microscopic animals that survive desiccation by inducing biostasis. To survive drying tardigrades rely on intrinsically disordered CAHS proteins, which also function to prevent perturbations induced by drying in vitro and in heterologous systems. CAHS proteins have been shown to form gels both in vitro and in vivo, which has been speculated to be linked to their protective capacity. However, the sequence features and mechanisms underlying gel formation and the necessity of gelation for protection have not been demonstrated. Here we report a mechanism of fibrillization and gelation for CAHS D similar to that of intermediate filament assembly. We show that in vitro, gelation restricts molecular motion, immobilizing and protecting labile material from the harmful effects of drying. In vivo, we observe that CAHS D forms fibrillar networks during osmotic stress. Fibrillar networking of CAHS D improves survival of osmotically shocked cells. We observe two emergent properties associated with fibrillization; (i) prevention of cell volume change and (ii) reduction of metabolic activity during osmotic shock. We find that there is no significant correlation between maintenance of cell volume and survival, while there is a significant correlation between reduced metabolism and survival. Importantly, CAHS D's fibrillar network formation is reversible and metabolic rates return to control levels after CAHS fibers are resolved. This work provides insights into how tardigrades induce reversible biostasis through the self-assembly of labile CAHS gels.

Storage cell proliferation during somatic growth establishes that tardigrades are not eutelic organisms.

Tardigrades, microscopic ecdysozoans known for extreme environment resilience, were traditionally believed to maintain a constant cell number after completing embryonic development, a phenomenon termed eutely. However, sporadic reports of dividing cells have raised questions about this assumption. In this study, we explored tardigrade post-embryonic cell proliferation using the model species Hypsibius exemplaris. Comparing hatchlings to adults, we observed an increase in the number of storage cells, responsible for nutrient storage. We monitored cell proliferation via 5-ethynyl-2'-deoxyuridine (EdU) incorporation, revealing large numbers of EdU+ storage cells during growth, which starvation halted. EdU incorporation associated with molting, a vital post-embryonic development process involving cuticle renewal for further growth. Notably, DNA replication inhibition strongly reduced EdU+ cell numbers and caused molting-related fatalities. Our study is the first to demonstrate using molecular approaches that storage cells actively proliferate during tardigrade post-embryonic development, providing a comprehensive insight into replication events throughout their somatic growth. Additionally, our data underscore the significance of proper DNA replication in tardigrade molting and survival. This work definitely establishes that tardigrades are not eutelic, and offers insights into cell cycle regulation, replication stress, and DNA damage management in these remarkable creatures as genetic manipulation techniques emerge within the field.

Protective roles of highly conserved motif 1 in tardigrade cytosolic-abundant heat soluble protein in extreme environments.

Tardigrades are remarkable microscopic animals that survive harsh conditions such as desiccation and extreme temperatures. Tardigrade-specific intrinsically disordered proteins (TDPs) play an essential role in the survival of tardigrades in extreme environments. Cytosolic-abundant heat soluble (CAHS) protein, a key TDP, is known to increase desiccation tolerance and to protect the activity of several enzymes under dehydrated conditions. However, the function and properties of each CAHS domain have not yet been elucidated in detail. Here, we aimed to elucidate the protective role of highly conserved motif 1 of CAHS in extreme environmental conditions. To examine CAHS domains, three protein constructs, CAHS Full (1-229), CAHS ∆Core (1-120_184-229), and CAHS Core (121-183), were engineered. The highly conserved CAHS motif 1 (124-142) in the CAHS Core formed an amphiphilic α helix, reducing the aggregate formation and protecting lactate dehydrogenase activity during dehydration-rehydration and freeze-thaw treatments, indicating that CAHS motif 1 in the CAHS Core was essential for maintaining protein solubility and stability. Aggregation assays and confocal microscopy revealed that the intrinsically disordered N- and C-terminal domains were more prone to aggregation under our experimental conditions. By explicating the functions of each domain in CAHS, our study proposes the possibility of using engineered proteins or peptides derived from CAHS as a potential candidate for biological applications in extreme environmental stress responses.

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.

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.

Ecology explains anhydrobiotic performance across tardigrades, but the shared evolutionary history matters more.

Desiccation stress is lethal to most animals. However, some microinvertebrate groups have evolved coping strategies, such as the ability to undergo anhydrobiosis (i.e. survival despite the loss of almost all body water). Tardigrades are one such group, where the molecular mechanisms of anhydrobiosis have been more thoroughly studied. Despite the ecological, evolutionary and biotechnological importance of anhydrobiosis, little is known about its inter- and intra-specific variability nor its relationship with natural habitat conditions or phylogenetic history. We developed a new index-anhydrobiotic recovery index (ARI)-to evaluate the anhydrobiotic performance of tardigrade populations from the family Macrobiotidae. Moreover, we compared the explanatory role of habitat humidity and phylogenetic history on this trait using a variance partitioning approach. We found that ARI is correlated with both microhabitat humidity and yearly rainfall, but it is mostly driven by phylogenetic niche conservatism (i.e. a high portion of ARI variation is explained by phylogeny alone). Finally, we showed that anhydrobiotic performance is highly variable, even between closely related species, and that their response to local ecological conditions is tightly linked to their phylogenetic history. This study not only presents key insights into an emerging model system, but also provides a new methodological approach for wider scale studies of the ecological and evolutionary implications of anhydrobiosis.

A new, simplified, drying protocol to prepare tardigrades for scanning electron microscopy.

A new protocol for preparation of tardigrades for scanning electron microscope (SEM) analysis is proposed. The more conventional protocols require various steps and a long time to obtain good drying of water bears, together with specific and uncommon instruments (i.e., critical point dryer) or highly volatile toxic compounds (i.e., hexametildisilazane). The new protocol can be performed using few and simple instruments and materials, all easily accessible, and produces a high yield in terms of dried animals in excellent condition for the observation of external morphological structures with SEM. The acquired data exhibit considerable promise, and the proposed methodology shows potential for application to other meiofaunal groups, including small arthropods, nematodes, and rotifers. RESEARCH HIGHLIGHTS: Cheap, safe, and fast new method for Tardigrada preparation for SEM. With the new protocol, the number of animals required for SEM studies is minimized. New protocol is potentially applicable to the study of other meiofaunal soft-bodied taxa.

The Evolution of Temperature and Desiccation-Related Protein Families in Tardigrada Reveals a Complex Acquisition of Extremotolerance.

Tardigrada is an ecdysozoan lineage famed for its resilience. Tardigrades can tolerate high doses of radiation, low-oxygen environments, desiccation, and both high and low temperatures under a dormant state called "anhydrobiosis", which is a reversible halt of metabolism upon almost complete desiccation. A large amount of research has focused on the genetic pathways related to these capabilities, and a number of genes have been identified and linked to the extremotolerant response of tardigrades. However, the history of these genes is unclear, and the origins and history of extremotolerant genes within Tardigrada remain a mystery. Here, we generate the first phylogenies of six separate protein families linked with desiccation and radiation tolerance in Tardigrada: cytosolic abundant heat-soluble protein, mitochondrial abundant heat-soluble protein, secretory abundant heat-soluble protein, meiotic recombination 11 homolog, and the newly discovered Echiniscus testudo abundant heat-soluble proteins (alpha and beta). The high number of independent gene duplications found amongst the six gene families studied suggests that tardigrades have a complex history with numerous independent adaptations to cope with aridity within the limnoterrestrial environment. Our results suggest that tardigrades likely transitioned from a marine environment to a limnoterrestrial environment only twice, once in stem Eutardigrada and once in Heterotardigrada, which explains the unique adaptations to anhydrobiosis present in both classes.

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.