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.

Cambrian lobopodians shed light on the origin of the tardigrade body plan.

Phylum Tardigrada (water bears), well known for their cryptobiosis, includes small invertebrates with four paired limbs and is divided into two classes: Eutardigrada and Heterotardigrada. The evolutionary origin of Tardigrada is known to lie within the lobopodians, which are extinct soft-bodied worms with lobopodous limbs mostly discovered at sites of exceptionally well-preserved fossils. Contrary to their closest relatives, onychophorans and euarthropods, the origin of morphological characters of tardigrades remains unclear, and detailed comparison with the lobopodians has not been well explored. Here, we present detailed morphological comparison between tardigrades and Cambrian lobopodians, with a phylogenetic analysis encompassing most of the lobopodians and three panarthropod phyla. The results indicate that the ancestral tardigrades likely had a Cambrian lobopodian-like morphology and shared most recent ancestry with the luolishaniids. Internal relationships within Tardigrada indicate that the ancestral tardigrade had a vermiform body shape without segmental plates, but possessed cuticular structures surrounding the mouth opening, and lobopodous legs terminating with claws, but without digits. This finding is in contrast to the long-standing stygarctid-like ancestor hypothesis. The highly compact and miniaturized body plan of tardigrades evolved after the tardigrade lineage diverged from an ancient shared ancestor with the luolishaniids.

In vivo expression vector derived from anhydrobiotic tardigrade genome enables live imaging in Eutardigrada.

Water is essential for life, but anhydrobiotic tardigrades can survive almost complete dehydration. Anhydrobiosis has been a biological enigma for more than a century with respect to how organisms sustain life without water, but the few choices of genetic toolkits available in tardigrade research have been a challenging circumstance. Here, we report the development of an in vivo expression system for tardigrades. This transient transgenic technique is based on a plasmid vector (TardiVec) with promoters that originated from an anhydrobiotic tardigrade Ramazzottius varieornatus. It enables the introduction of GFP-fused proteins and genetically encoded indicators such as the Ca2+ indicator GCaMP into tardigrade cells; consequently, the dynamics of proteins and cells in tardigrades may be observed by fluorescence live imaging. This system is applicable for several tardigrades in the class Eutardigrada: the promoters of anhydrobiosis-related genes showed tissue-specific expression in this work. Surprisingly, promoters functioned similarly between multiple species, even for species with different modes of expression of anhydrobiosis-related genes, such as Hypsibius exemplaris, in which these genes are highly induced upon facing desiccation, and Thulinius ruffoi, which lacks anhydrobiotic capability. These results suggest that the highly dynamic expression changes in desiccation-induced species are regulated in trans. Tissue-specific expression of tardigrade-unique unstructured proteins also suggests differing anhydrobiosis machinery depending on the cell types. We believe that tardigrade transgenic technology opens up various experimental possibilities in tardigrade research, especially to explore anhydrobiosis mechanisms.

Stress-dependent cell stiffening by tardigrade tolerance proteins that reversibly form a filamentous network and gel.

Tardigrades are able to tolerate almost complete dehydration by entering a reversible ametabolic state called anhydrobiosis and resume their animation upon rehydration. Dehydrated tardigrades are exceptionally stable and withstand various physical extremes. Although trehalose and late embryogenesis abundant (LEA) proteins have been extensively studied as potent protectants against dehydration in other anhydrobiotic organisms, tardigrades produce high amounts of tardigrade-unique protective proteins. Cytoplasmic-abundant heat-soluble (CAHS) proteins are uniquely invented in the lineage of eutardigrades, a major class of the phylum Tardigrada and are essential for their anhydrobiotic survival. However, the precise mechanisms of their action in this protective role are not fully understood. In the present study, we first postulated the presence of tolerance proteins that form protective condensates via phase separation in a stress-dependent manner and searched for tardigrade proteins that reversibly form condensates upon dehydration-like stress. Through a comprehensive search using a desolvating agent, trifluoroethanol (TFE), we identified 336 proteins, collectively dubbed "TFE-Dependent ReversiblY condensing Proteins (T-DRYPs)." Unexpectedly, we rediscovered CAHS proteins as highly enriched in T-DRYPs, 3 of which were major components of T-DRYPs. We revealed that these CAHS proteins reversibly polymerize into many cytoskeleton-like filaments depending on hyperosmotic stress in cultured cells and undergo reversible gel-transition in vitro. Furthermore, CAHS proteins increased cell stiffness in a hyperosmotic stress-dependent manner and counteract the cell shrinkage caused by osmotic pressure, and even improved the survival against hyperosmotic stress. The conserved putative helical C-terminal region is necessary and sufficient for filament formation by CAHS proteins, and mutations disrupting the secondary structure of this region impaired both the filament formation and the gel transition. On the basis of these results, we propose that CAHS proteins are novel cytoskeleton-like proteins that form filamentous networks and undergo gel-transition in a stress-dependent manner to provide on-demand physical stabilization of cell integrity against deformative forces during dehydration and could contribute to the exceptional physical stability in a dehydrated state.

Tardigrades Use Intrinsically Disordered Proteins to Survive Desiccation.

Tardigrades are microscopic animals that survive a remarkable array of stresses, including desiccation. How tardigrades survive desiccation has remained a mystery for more than 250 years. Trehalose, a disaccharide essential for several organisms to survive drying, is detected at low levels or not at all in some tardigrade species, indicating that tardigrades possess potentially novel mechanisms for surviving desiccation. Here we show that tardigrade-specific intrinsically disordered proteins (TDPs) are essential for desiccation tolerance. TDP genes are constitutively expressed at high levels or induced during desiccation in multiple tardigrade species. TDPs are required for tardigrade desiccation tolerance, and these genes are sufficient to increase desiccation tolerance when expressed in heterologous systems. TDPs form non-crystalline amorphous solids (vitrify) upon desiccation, and this vitrified state mirrors their protective capabilities. Our study identifies TDPs as functional mediators of tardigrade desiccation tolerance, expanding our knowledge of the roles and diversity of disordered proteins involved in stress tolerance.

The embryonic origin of primordial germ cells in the tardigrade Hypsibius exemplaris.

Primordial germ cells (PGCs) give rise to gametes - cells necessary for the propagation and fertility of diverse organisms. Current understanding of PGC development is limited to the small number of organisms whose PGCs have been identified and studied. Expanding the field to include little-studied taxa and emerging model organisms is important to understand the full breadth of the evolution of PGC development. In the phylum Tardigrada, no early cell lineages have been identified to date using molecular markers. This includes the PGC lineage. Here, we describe PGC development in the model tardigrade Hypsibius exemplaris. The four earliest-internalizing cells (EICs) exhibit PGC-like behavior and nuclear morphology. The location of the EICs is enriched for mRNAs of conserved PGC markers wiwi1 (water bear piwi 1) and vasa. At early stages, both wiwi1 and vasa mRNAs are detectable uniformly in embryos, which suggests that these mRNAs do not serve as localized determinants for PGC specification. Only later are wiwi1 and vasa enriched in the EICs. Finally, we traced the cells that give rise to the four PGCs. Our results reveal the embryonic origin of the PGCs of H. exemplaris and provide the first molecular characterization of an early cell lineage in the tardigrade phylum. We anticipate that these observations will serve as a basis for characterizing the mechanisms of PGC development in this animal.

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.

Pinpointing the microbiota of tardigrades: What is really there?

Microbiota are considered significant in the biology of tardigrades, yet their diversity and distribution remain largely unexplored. This is partly due to the methodological challenges associated with studying the microbiota of small organisms that inhabit microbe-rich environments. In our study, we characterized the microbiota of 31 species of cultured tardigrades using 16S rRNA amplicon sequencing. We employed various sample preparation strategies and multiple types of controls and estimated the number of microbes in samples using synthetic DNA spike-ins. We also reanalysed data from previous tardigrade microbiome studies. Our findings suggest that the microbial communities of cultured tardigrades are predominantly composed of bacterial genotypes originating from food, medium, or reagents. Despite numerous experiments, we found it challenging to identify strains that were enriched in certain tardigrades, which would have indicated likely symbiotic associations. Putative tardigrade-associated microbes rarely constituted more than 20% of the datasets, although some matched symbionts identified in other studies. We also uncovered serious contamination issues in previous tardigrade microbiome studies, casting doubt on some of their conclusions. We concluded that tardigrades are not universally dependent on specialized microbes. Our work underscores the need for rigorous safeguards in studies of the microbiota of microscopic organisms and serves as a cautionary tale for studies involving samples with low microbiome abundance.

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.

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.

Tardigrades exhibit robust interlimb coordination across walking speeds and terrains.

Tardigrades must negotiate heterogeneous, fluctuating environments and accordingly utilize locomotive strategies capable of dealing with variable terrain. We analyze the kinematics and interleg coordination of freely walking tardigrades (species: Hypsibius exemplaris). We find that tardigrade walking replicates several key features of walking in insects despite disparities in size, skeleton, and habitat. To test the effect of environmental changes on tardigrade locomotor control circuits we measure kinematics and interleg coordination during walking on two substrates of different stiffnesses. We find that the phase offset between contralateral leg pairs is flexible, while ipsilateral coordination is preserved across environmental conditions. This mirrors similar results in insects and crustaceans. We propose that these functional similarities in walking coordination between tardigrades and arthropods is either due to a generalized locomotor control circuit common to panarthropods or to independent convergence onto an optimal strategy for robust multilegged control in small animals with simple circuitry. Our results highlight the value of tardigrades as a comparative system toward understanding the mechanisms-neural and/or mechanical-underlying coordination in panarthropod locomotion.

Transcriptome analysis of the tardigrade Hypsibius exemplaris exposed to the DNA-damaging agent bleomycin.

Tardigrades are microscopic animals that are renowned for their capabilities of tolerating near-complete desiccation by entering an ametabolic state called anhydrobiosis. However, many species also show high tolerance against radiation in the active state as well, suggesting cross-tolerance via the anhydrobiosis mechanism. Previous studies utilized indirect DNA damaging agents to identify core components of the cross-tolerance machinery in species with high anhydrobiosis capacities. However, it was difficult to distinguish whether transcriptomic changes were specific to DNA damage or mutual with anhydrobiosis. To this end, we performed transcriptome analysis on bleomycin-exposed Hypsibius exemplaris. We observed induction of several tardigrade-specific gene families, including a previously identified novel anti-oxidative stress family, which may be a core component of the cross-tolerance mechanism. We also identified enrichment of the tryptophan metabolism pathway, for which metabolomic analysis suggested engagement of this pathway in stress tolerance. These results provide several candidates for the core component of cross-tolerance, as well as possible anhydrobiosis machinery.

Antioxidant Defense in the Toughest Animals on the Earth: Its Contribution to the Extreme Resistance of Tardigrades.

Tardigrades are unique among animals in their resistance to dehydration, mainly due to anhydrobiosis and tun formation. They are also very resistant to high-energy radiation, low and high temperatures, low and high pressure, and various chemical agents, Interestingly, they are resistant to ionizing radiation both in the hydrated and dehydrated states to a similar extent. They are able to survive in the cosmic space. Apparently, many mechanisms contribute to the resistance of tardigrades to harmful factors, including the presence of trehalose (though not common to all tardigrades), heat shock proteins, late embryogenesis-abundant proteins, tardigrade-unique proteins, DNA repair proteins, proteins directly protecting DNA (Dsup and TDR1), and efficient antioxidant system. Antioxidant enzymes and small-molecular-weight antioxidants are an important element in the tardigrade resistance. The levels and activities of many antioxidant proteins is elevated by anhydrobiosis and UV radiation; one explanation for their induction during dehydration is provided by the theory of "preparation for oxidative stress", which occurs during rehydration. Genes coding for some antioxidant proteins are expanded in tardigrades; some genes (especially those coding for catalases) were hypothesized to be of bacterial origin, acquired by horizontal gene transfer. An interesting antioxidant protein found in tardigrades is the new Mn-dependent peroxidase.

Properties of a tardigrade desiccation-tolerance protein aerogel.

Lyophilization is promising for tackling degradation during the drying and storage of protein-based drugs. Tardigrade cytosolically abundant heat soluble (CAHS) proteins are necessary and sufficient for desiccation-tolerance in vivo and protein protection in vitro. Hydrated CAHS proteins form coiled-coil-based fine-stranded, cold-setting hydrogels, but the dried protein remains largely uncharacterized. Here, we show that dried CAHS D gels (i.e., aerogels) retain the structural units of their hydrogels, but the details depend on prelyophilization CAHS concentrations. Low concentration samples (<10 g/L) form thin (<0.2 µm) tangled fibrils lacking regular structure on the micron scale. Upon increasing the concentration, the fibers thicken and form slabs comprising the walls of the aerogel pores. These changes in morphology are associated with a loss in disorder and an increase in large ß sheets and a decrease in α helices and random coils. This disorder-to-order transition is also seen in hydrated gels as a function of concentration. These results suggest a mechanism for pore formation and indicate that using CAHS proteins as excipients will require attention to initial conditions because the starting concentration impacts the lyophilized product.

Two new tardigrade genera from New Zealand's Southern Alp glaciers display morphological stasis and parallel evolution.

Tardigrada is an invertebrate phylum that often constitutes a dominant micrometazoan group on glaciers worldwide. We investigated tardigrades residing in surface ice above the equilibrium line altitude (ELA) on three temperate glaciers of New Zealand's Southern Alps. Morphological, morphometric and multilocus DNA analyses (CO1, 18S rRNA, 28S rRNA, ITS-2) revealed two new genera comprising four species, of which two are formally described here: Kopakaius gen. nov. nicolae sp. nov. and Kararehius gen. nov. gregorii sp. nov. The former is represented by three genetically distinct phyletic lineages akin to species. According to CO1, Kopakaius gen. nov. nicolae sp. nov. inhabits Whataroa Glacier only while the remaining two Kopakaius species occur on Fox and Franz Joseph Glaciers, suggesting low dispersal capabilities. Although morphological characteristics of the new genera could indicate affinity with the subfamily Itaquasconinae, phylogenetic analysis placed them confidently in the subfamily Diphasconinae. Kopakaius gen. nov. lack placoids in the pharynx similar with some Itaquasconinae, whereas dark pigmentation and claw shape aligns them with the glacier-obligate genus, Cryobiotus (subfamily Hypsibiinae), which is an example of parallel evolution. The second genus, Kararehius gen nov. could be classified as Adropion-like (subfamily Itaquasconinae), but differs greatly by genetics (placed in the subfamily Diphasconinae) as well as morphology (e.g., lack of septulum), exemplify deep stasis in Hypsibiidae. Our results suggest that glacier fragmentation during the Pleistocene triggered tardigrade speciation, making it a suitable model for studies on allopatric divergence in glacier meiofauna.

Expanding Acutuncus: Phylogenetics and morphological analyses reveal a considerably wider distribution for this tardigrade genus.

The tardigrade genus Acutuncus has been long thought to be an Antarctic endemism, well adapted to this harsh environment. The Antarctic endemicity of Acutuncus was recently dispelled with the description of Acutuncus mariae Zawierucha, 2020 found in the Svalbard archipelago. The integrated analyses on two newly found Acutuncus populations from UK and Italy, and a population of Acutuncus antarcticus found close to its type locality allowed us to expand the climatic and geographic range of the genus Acutuncus. These findings also allowed us to re-evaluate the morphological diagnoses of Acutuncus and accommodate it in the newly proposed monotypic family Acutuncidae fam. nov. Two new Acutuncus species morpho-groups are instituted based on eggs morphology: one (Acutuncus antarcticus morphogroup) including the Antarctic Acutuncus taxa characterized by eggs with long pillars within the chorion and eggs laid freely to the environment, the other (Acutuncus mariae morphogroup) including the European species, characterized by eggs with short pillars within the chorion and eggs laid in the exuvium. Finally, we describe two new Acutuncus species from Europe: Acutuncus mecnuffisp. nov. and Acutuncus giovanniniaesp. nov.

First evidence of sex-specific responses to chemical cues in tardigrade mate searching behaviour.

Chemical cues are widely used in intraspecific and interspecific communication, either as substances deposited in the substrate or as molecules diffused in water or air. In tardigrades, an emerging microscopic study system, chemical communication and its role in reproduction are poorly known. Here, we assessed sex differences in the detection of (a) short-range diffusing signals and (b) deposited cue trails during the mate-searching behaviour of freely moving virgin male and female Macrobiotus polonicus. We tracked individual behaviour (a) in simultaneous double-choice chambers, where live conspecifics of each sex were presented in water and (b) of freely moving pairs on agar without water. We found that males, but not females, preferentially associated with opposite-sex individuals in trials conducted in water. In contrast, neither sex detected nor followed cues deposited on agar. In conclusion, our study suggests that mate discrimination and approach are male-specific traits and are limited to waterborne chemical cues. These results support the existence of Darwinian sex roles in pre-mating behaviour in an animal group with virtually non-existing sex differences in morphology or ecology.

The unbearable uncertainty of panarthropod relationships.

Panarthropoda, the clade comprising the phyla Onychophora, Tardigrada and Euarthropoda, encompasses the largest majority of animal biodiversity. The relationships among the phyla are contested and resolution is key to understanding the evolutionary assembly of panarthropod bodyplans. Molecular phylogenetic analyses generally support monophyly of Onychophora and Euarthropoda to the exclusion of Tardigrada (Lobopodia hypothesis), which is also supported by some analyses of morphological data. However, analyses of morphological data have also been interpreted to support monophyly of Tardigrada and Euarthropoda to the exclusion of Onychophora (Tactopoda hypothesis). Support has also been found for a clade of Onychophora and Tardigrada that excludes Euarthropoda (Protarthropoda hypothesis). Here we show, using a diversity of phylogenetic inference methods, that morphological datasets cannot discriminate statistically between the Lobopodia, Tactopoda and Protarthropoda hypotheses. Since the relationships among the living clades of panarthropod phyla cannot be discriminated based on morphological data, we call into question the accuracy of morphology-based phylogenies of Panarthropoda that include fossil species and the evolutionary hypotheses based upon them.

A New Species of Bisexual Milnesium (Eutardigrada: Apochela) Having Aberrant Claws from Innhovde, Dronning Maud Land, East Antarctica.

There have been several records in the last 60 years for East Antarctica for Milnesium tardigradum Doyère, 1840 sensu lato, now considered a species complex. During the 56th Japanese Antarctic Research Expedition summer operation (2014-2015), a new tardigrade species in the genus Milnesium Doyère, 1840 was found in an ice-free Innhovde area along Lützow-Holm Bay, Dronning Maud Land, East Antarctica. The new species has aberrant claws with four to seven points on each secondary claw branch, which distinguishes it from other Milnesium species. A male specimen was found in the population and evidence showed that an isolated adult female moulted twice without oviposition. This strongly suggested bisexual reproduction for this population. The new species, Milnesium rastrum sp. nov., is described with its phylogenetic position and a discussion on the reproductive strategies for the harsh environments.