Reproduction of Mesobiotus: Comparison of Morphology and Behavior in the Family Macrobiotidae (Tardigrada: Eutardigrada).

The genus Mesobiotus was separated from the genus Macrobiotus in 2016 and the name referred to its phylogenetic position among the family Macrobiotidae; however, knowledge of the reproductive behavior of this genus is limited compared to those of Paramacrobiotus and Macrobiotus. This study comprehensively provides the reproductive traits, including the gamete morphologies and behavioral observations, of Mesobiotus. The morphology of its spermatozoon showed a length that was intermediary among those of Paramacrobiotus and Macrobiotus species. The sequence of mating behavior was generally conserved in the three species of Macrobiotidae. They showed the described five steps observed in Paramacrobiotus and Macrobiotus; however, the males of Mesobiotus repeated ejaculations in a mating session, which is the first observation of premature ejaculation in tardigrades. Our results indicated that Mesobiotus has the potential to be a model to show the linkage between genera with respect to the morphology and behavior in the family Macrobiotidae.

Spermatozoa morphology changes during reproduction and first observation of acrosomal contact in two dioecious species of Macrobiotidae (Tardigrada: Eutardigrada).

Mating behaviours for two species of dioecious eutardigrades: a strain of Paramacrobiotus sp. and Macrobiotus shonaicus (Stec et al., 2018) have been recorded previously, and observations have indicated that spermatozoa of both species are first released into the environment, then swim through the cloaca of the females and into the spermatheca. The fusion of gamete nuclei has not yet occurred in a laid egg. Therefore, it has been suggested that fertilization is completed externally as the egg is released into the environment before the nuclei of the gametes fuse. In the present study, the spermatozoa of both Paramacrobiotus sp. and M. shonaicus spermatozoa underwent morphological changes during reproduction. In morphometrical analyses of testicular spermatozoa, the tail, mid-piece, nucleus, and acrosome were significantly longer in Paramacrobiotus sp. compared with M. shonaicus. The nuclei of both the testicular and spermathecal spermatozoa were equally coiled, but the latter had shorter tails in both species. These spermatozoa were present on the surface of the egg chorion after oviposition. The tip of the acrosomes lay buried in the chorion, suggesting that penetration had occurred. We also proposed that the reduced tail is a conserved trait, at least in Macrobiotidae.

In Silico Reconstruction of Sperm Chemotaxis.

In echinoderms, sperm swims in random circles and turns in response to a chemoattractant. The chemoattractant evokes transient Ca2+ influx in the sperm flagellum and induces turning behavior. Recently, the molecular mechanisms and biophysical properties of this sperm response have been clarified. Based on these experimental findings, in this study, we reconstructed a sperm model in silico to demonstrate an algorithm for sperm chemotaxis. We also focused on the importance of desensitizing the chemoattractant receptor in long-range chemotaxis because sperm approach distantly located eggs, and they must sense the chemoattractant concentration over a broad range. Using parameters of the sea urchin, simulations showed that a number of sperm could reach the egg from millimeter-order distances with desensitization, indicating that we could organize a functional sperm model, and that desensitization of the receptor is essential for sperm chemotaxis. Then, we compared the model with starfish sperm, which has a different desensitization scheme and analyzed the properties of the model against various disturbances. Our approach can be applied as a novel tool in chemotaxis research.

Milnesium (Tardigrada: Apochela) in Japan: The First Confirmed Record of Milnesium tardigradum s.s. and Description of Milnesium pacificum sp. nov.

Presently, more than 40 species of the genus Milnesium Doyère, 1840 (Tardigrada: Eutardigrada: Apochela: Milnesiidae) have been described. In Japan, however, almost all records of milnesiid tardigrades should be re-examined with the current criteria on the taxonomy of this genus, except for one species, the recently described Milnesium inceptum Morek, Suzuki, Schill, Georgiev, Yankova, Marley, and Michalczyk, 2019. In this study, we found two species, Milnesium pacificum sp. nov. and Milnesium tardigradum Doyère, 1840, from three southern islands and two cold regions in Japan, respectively. Milnesium pacificum sp. nov., having dorsal sculpturing, exhibits an early positive change in claw configuration. On the other hand, M. tardigradum s.s. from Japan has an early negative claw configuration change, as has been reported in a recent study on the neotype population of this species. We performed DNA barcoding for both species, which indicated that M. pacificum sp. nov. has a close affinity with an undescribed Milnesium species collected from Brazil, and that M. tardigradum from Japan represents the recently described subclade that contains specimens from Poland, Hungary, and Russia. The chromosome numbers were 2n = 14 in M. pacificum sp. nov. and 2n = 10 in M. tardigradum. We detected at least three species of the genus Milnesium present in Japan. Our results advance the investigation of the relationship between phylogenetic position and characteristic morphology as well as expand the known geographic range of M. tardigradum.

Comparison of the transcriptomes of two tardigrades with different hatching coordination.

BACKGROUND: Tardigrades 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. RESULTS: To 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. CONCLUSION: These observations suggest a hatching regulation mechanism by the molting pathway in H. exemplaris.

Comparison of Sexual Reproductive Behaviors in Two Species of Macrobiotidae (Tardigrada: Eutardigrada).

Reproductive strategy is an important aspect of biological diversity. In tardigrades, several reproductive modes, including sexual reproduction, are known. However, tardigrade mating behavior has been observed only rarely in most species, and in some cases, especially in the freely ovipositing eutardigrades, remains entirely unknown. In the present study, we cultured two sexually reproducing tardigrade species that lay eggs freely, Paramacrobiotus sp. TYO strain and Macrobiotus shonaicus, to investigate and compare their courtship, mating, and chromosome morphology. Mating behavior was observed and recorded in both species. The entire mating sequence, including courtship, was categorized into five discrete steps common to two species, as follows: [1] Tracking: the male tracks and orientates toward the female; [2] Touching: the male makes contact with the cloaca of the female; [3] Standstill: the female ceases movement until male ejaculation is complete; [4] Ejaculation: the male curls its caudal end and ejaculates into the cloaca from close range; [5] Contraction: the female contracts its ventral side after ejaculation to capture spermatozoa deposited in the external environment in close proximity to the cloaca. Some notable differences between the two species were observed in the steps 3-4. First, oviposition was observed at 40 min in Paramacrobiotus sp. TYO strain, and a few days after mating in M. shonaicus, respectively. Comparisons of chromosome morphology before and after mating indicated that oocytes are arrested at metaphase I in both species. Spermatozoa attach to the interior of the chorion of laid eggs.

Inhibition of Dr-dut gene causes DNA damage in planarian.

The DUT gene encodes Deoxyuridine triphosphatase (dUTPase), which is involved in nucleotide metabolism. dUTPase prevents uracil misincorporation in DNA by balancing the intracellular ratio between dUTP and dTTP. This study aimed to investigate the role of Dr-dut gene in the planarian Dugesia ryukyuensis by assessing the consequences of Dr-dut silencing on known phenomena, including regeneration following amputation and radiation damage. We functionally disrupted planarian Dr-dut mRNA by feeding RNAi-containing food to animals. Dr-dut RNAi resulted in the death of planarians in 28 days, and elevated double-stranded DNA breakage. Expression of the DNA damage response gene Dr-atm and the DNA repair genes Dr-rad51 and Dr-rad51c temporarily increased, and then decreased following the onset of feeding. When RNAi-treated planarians were amputated, both head and tail parts failed to regenerate, and the animals died in 25 and 29 days, respectively. Administration of 5-fluorouracil (5-FU) also resulted in death and DNA damage, and synergistically caused higher genotoxicity in planarian fed Dr-dut RNAi-containing food.

Protein kinase A activity leads to the extension of the acrosomal process in starfish sperm.

Acrosomal vesicles (AVs) of sperm undergo exocytosis during the acrosome reaction, which is immediately followed by the actin polymerization-dependent extension of an acrosomal process (AP) in echinoderm sperm. In the starfish Asterias amurensis, a large proteoglycan, acrosome reaction-inducing substance (ARIS), together with asteroidal sperm-activating peptide (asterosap) and/or cofactor for ARIS, induces the acrosome reaction. Asterosap induces a transient elevation of intracellular cGMP and Ca2+ levels, and, together with ARIS, causes a sustained increase in intracellular cAMP and Ca2+ . Yet, the contribution of signaling molecules downstream of cAMP and Ca2+ in inducing AV exocytosis and AP extension remain unknown. A modified acrosome reaction assay was used here to differentiate between AV exocytosis and AP extension in starfish sperm, leading to the discovery that Protein kinase A (PKA) inhibitors block AP extension but not AV exocytosis. Additionally, PKA-mediated phosphorylation of target proteins occurs, and these substrates localize at the base of the AP, demonstrating that PKA activation regulates an AP extension step during the acrosome reaction. The major PKA substrate was further identified, from A. amurensis and Asterias forbesi sperm, as a novel protein containing six PKA phosphorylation motifs. This protein, referred to as PKAS1, likely plays a key role in AP actin polymerization during the acrosome reaction.

Triploid planarian reproduces truly bisexually with euploid gametes produced through a different meiotic system between sex.

Although polyploids are common among plants and some animals, polyploidization often causes reproductive failure. Triploids, in particular, are characterized by the problems of chromosomal pairing and segregation during meiosis, which may cause aneuploid gametes and results in sterility. Thus, they are generally considered to reproduce only asexually. In the case of the Platyhelminthes Dugesia ryukyuensis, populations with triploid karyotypes are normally found in nature as both fissiparous and oviparous triploids. Fissiparous triploids can also be experimentally sexualized if they are fed sexual planarians, developing both gonads and other reproductive organs. Fully sexualized worms begin reproducing by copulation rather than fission. In this study, we examined the genotypes of the offspring obtained by breeding sexualized triploids and found that the offspring inherited genes from both parents, i.e., they reproduced truly bisexually. Furthermore, meiotic chromosome behavior in triploid sexualized planarians differed significantly between male and female germ lines, in that female germ line cells remained triploid until prophase I, whereas male germ line cells appeared to become diploid before entry into meiosis. Oocytes at the late diplotene stage contained not only paired bivalents but also unpaired univalents that were suggested to produce diploid eggs if they remained in subsequent processes. Triploid planarians may therefore form euploid gametes by different meiotic systems in female and male germ lines and thus are be able to reproduce sexually in contrast to many other triploid organisms.

DrRad51 is required for chiasmata formation in meiosis in planarian Dugesia ryukyuensis.

Rad51, a conserved eukaryotic protein, mediates the homologous-recombination repair of DNA double-strand breaks that occur during both mitosis and meiosis. During prophase I of meiosis, homologous recombination enhances the linkage between homologous chromosomes to increase the accuracy of segregation at anaphase I. In polyploidy situations, however, difficulties with homologous chromosome segregation often disrupt meiosis. Yet, triploid individuals of the planarian Dugesia ryukyuensis are able to produce functional gametes through a specialized form of meiosis. To shed light on the molecular mechanisms that promote successful meiosis in triploid D. ryukyuensis, we investigated rad51 gene function. We isolated three genes of the Rad51 family, the Rad51 homolog Dr-rad51 and the Rad51 paralogs Dr-rad51B and Dr-rad51C. Dr-rad51 was expressed in germ-line and presumably in somatic stem cells, but was not necessary for the regeneration of somatic tissue. RNA-interference (RNAi) depletion of Dr-rad51 during sexualization did not affect chromosome behavior in zygotene oocytes, but did result in the loss of chiasmata at the diplotene stage. Thus, homologous recombination does not appear to be necessary for synapsis, but is needed for crossover and proper segregation in D. ryukyuensis.

Sexual dimorphism in the tardigrade Paramacrobiotus metropolitanus transcriptome.

BACKGROUND: In gonochoristic animals, the sex determination pathway induces different morphological and behavioral features that can be observed between sexes, a condition known as sexual dimorphism. While many components of this sex differentiation cascade show high levels of diversity, factors such as the Doublesex-Mab-3-Related Transcription factor (DMRT) are widely conserved across animal taxa. Species of the phylum Tardigrada exhibit remarkable diversity in morphology and behavior between sexes, suggesting a pathway regulating this dimorphism. Despite the wealth of genomic and zoological knowledge accumulated in recent studies, the sexual differences in tardigrades genomes have not been identified. In the present study, we focused on the gonochoristic species Paramacrobiotus metropolitanus and employed omics analyses to unravel the molecular basis of sexual dimorphism. RESULTS: Transcriptome analysis between sex-identified specimens revealed numerous differentially expressed genes, of which approximately 2,000 male-biased genes were focused on 29 non-male-specific genomic loci. From these regions, we identified two Macrobiotidae family specific DMRT paralogs, which were significantly upregulated in males and lacked sex specific splicing variants. Furthermore, phylogenetic analysis indicated all tardigrade genomes lack the doublesex ortholog, suggesting doublesex emerged after the divergence of Tardigrada. In contrast to sex-specific expression, no evidence of genomic differences between the sexes was found. We also identified several anhydrobiosis genes that exhibit sex-biased expression, suggesting a possible mechanism for protection of sex-specific tissues against extreme stress. CONCLUSIONS: This study provides a comprehensive analysis for analyzing the genetic differences between sexes in tardigrades. The existence of male-biased, but not male-specific, genomic loci and identification of the family specific male-biased DMRT subfamily provides the foundation for understanding the sex determination cascade. In addition, sex-biased expression of several tardigrade-specific genes which are involved their stress tolerance suggests a potential role in protecting sex-specific tissue and gametes.

Drpiwi-1 is essential for germline cell formation during sexualization of the planarian Dugesia ryukyuensis.

A piwi homolog is required for the regulation of stem cells, formation and maintenance of germline stem cells, and gametogenesis in many metazoans. Planarians can change their reproductive mode seasonally, both asexually and sexually, and develop and maintain germ cells and sexual organs. They have many pluripotent stem cells (neoblasts) that can differentiate into both somatic and germline stem cells. Thus, we searched for a piwi subfamily in the planarian Dugesia ryukyuensis. Four piwi homologs, identified as Drpiwi-1, -2, -3, and -4, were expressed in sexually reproductive worms. We then selectively destroyed the neoblasts by irradiating the worms with X-rays. In such worms, Drpiwi-1, -2, and -3 were not expressed at all, whereas Drpiwi-4 was expressed to the same degree as that in non-irradiated controls, indicating that Drpiwi-1, -2, and -3, but not Drpiwi-4, are expressed in neoblasts. During the regeneration process, Drpiwi-2(RNAi) and -3(RNAi) worms failed to regenerate after ablation, but Drpiwi-1 and -4(RNAi) worms regenerated. During the sexualizing process, Drpiwi-1(RNAi) worms failed to develop ovaries and testes, but somatic sexual organs were unaffected. Germ cell development was normal in Drpiwi-4(RNAi) worms. Therefore, Drpiwi-2 and -3 may be related to the regulation of neoblasts important for maintaining homeostasis, and Drpiwi-1 is essential for the development of germ cells but not somatic sexual organs. DrPiwi-1 is localized in the cytoplasm of stem cells and germline cells and may be involved in regulating some gene expression. We suggest that planarian Piwi controls germline formation via RNA silencing mechanisms.

Neuropeptides trigger oocyte maturation and subsequent spawning in the hydrozoan jellyfish Cytaeis uchidae.

Oocyte maturation and subsequent spawning in hydrozoan jellyfish are generally triggered by light-dark cycles. To examine if the initiation of the maturation process after light stimulus is mediated by neurotransmitters, neuropeptides isolated originally from Hydra magnipapillata were applied to sexually mature female medusae of the hydrozoan jellyfish Cytaeis uchidae. Among the Hydra neuropeptides tested, Hym-53 (NPYPGLW-NH2 ), as well as a nonphysiological peptide, CGLWamide (CGLW-NH2 ), were most effective in inducing oocyte maturation and spawning. Hym-355 (FPQSFLPRG-NH2 ) also triggered these events, but the stimulatory effect was weaker. Since Hym-53-OH (NPYPGLW) and Hym-355-OH (FPQSFLPRG) had no effect, amidation at the C-terminus may be critical for the stimulatory activities of the peptides. Exposure to Hym-53 for 2 min was sufficient to trigger of oocyte maturation, and the spawned eggs were able to be fertilized and to develop normally. Transmission electron microscopy confirmed that bundles of axon-like structures that contain dense-core synaptic vesicles and microtubules are present in the ovarian ectodermal epithelium overlying the oocytes. In addition, immunohistological analyses revealed that some of the neurons in the ectodermal epithelium are GLWamide- and PRGamide-positive. These results suggest that a neuropeptide signal transduction pathway is involved in mediating the induction of oocyte maturation and spawning in this jellyfish.

Structure of acrosome reaction-inducing substance in the jelly coat of starfish eggs: a mini review.

Our knowledge at present on the structure of acrosome-reaction inducing substance (ARIS) in the jelly coat of starfish eggs is summarized. ARIS ia a proteoglycan-like molecule consisting of very long, linear, and highly sulfated glycans and three ARIS proteins, ARIS1-3. Detailed structures of the major glycan of ARIS and of ARIS1-3 are discussed. 3D-models of ARIS glycans are also presented. Phylogenetic distribution of ARIS proteins and/or genes indicates that ARIS genes are well preserved from the Ctenophore to Cephalochordata. In the Echinodermata, ARIS1-3 and ARIS genes were detected in all classes except for sea urchins.

Starfish ApDOCK protein essentially functions in larval defense system operated by mesenchyme cells.

In larvae of the starfish, Asterina pectinifera, mesenchyme cells operate in the defense system through various behaviors. We have investigated mesenchyme cell dynamics during the immune response by identifying ApDOCK, a new member of the DOCK180 superfamily protein. In 4-day-old bipinnaria larvae processed for morpholino oligonucleotide-mediated knockdown of ApDOCK, injection of inorganic foreign substances revealed that (1) mesenchyme cells fail to undergo either directed migration toward a large oil-droplet or persistent spreading on the oil-droplet after contact; (2) neither uptake of micro-beads nor cell-to-cell fusion on the large oil-droplet differed from that of mesenchyme cells from control larvae. Similar behaviors were also recorded in experiments where bacteria were injected. Under culture conditions, the expression level of ApDOCK mRNA was significantly associated with the immunological behavior of mesenchyme cells. Apparently, the mesenchyme cells from ApDOCK loss-of-function larvae exhibited insufficient lamellipodium formation via lack of fibrous form of actin organization at the leading edge. These results suggest that the migratory congregation and persistence of encapsulation of larval mesenchyme cells are intracellularly regulated by ApDOCK protein, and this regulation is associated with organization of cytoskeletal actin.

Reproductive mode and ovarian morphology regulation in chimeric planarians composed of asexual and sexual neoblasts.

Planarians are comprised of populations with different reproductive strategies: exclusively innately asexual (AS), exclusively innately sexual (InS), and seasonally switching. AS worms can be sexualized experimentally by feeding them with minced InS worms, and the resultant worms are characterized as acquired sexual (AqS). Differences between InS and AqS worms are expected to provide important clues to the poorly understood mechanism underlying the regulation of their reproductive mode. Morphological differences were found between InS and AqS worm ovaries, and we showed that the pluripotent stem cells (neoblasts) from InS worms, but not those of AqS worms, have the capacity to initiate the sexual state autonomously via neoblast fraction transplantation. To compare their reproductive mode and ovarian morphology regulation, InS donor neoblast fractions were transplanted into non-lethally X-ray-irradiated AS recipients. All transplants showed stable chimerism and reproduced sexually, suggesting that InS worm neoblasts can initiate sexual state autonomously, even when coexisting with AS worm neoblasts. The chimeras formed extraordinarily large and supernumerary ovaries equivalent to AqS worms, which were not seen in InS worms, suggesting that regulation of ovarian morphology in AS worm-derived cells in response to endogenous sexualizing stimulation distinctly differs from that of InS worms.

Stem cells from innate sexual but not acquired sexual planarians have the capability to form a sexual individual.

Planarian species may harbor as many as three populations with different reproductive strategies. Animals from innate asexual (AS) and innate sexual (InS) populations reproduce only by fission and cross-fertilization, respectively, whereas the third population switches seasonally between the two reproductive modes. AS worms can be experimentally sexualized by feeding them with minced InS worms; we termed the resulting animals "acquired sexual" (AqS) worms. Both AqS and InS worms exhibit sexualizing activity when used as feed, suggesting that they maintain their sexual state via endogenous sexualizing substances, although the mechanisms underlying determination of reproductive strategy and sexual switching in these metazoans remain enigmatic. Therefore, we compared the endogenous sexualizing activity of InS worms and AqS worms. First, we amputated mature worms and assessed if they could re-enter a sexual state. Regenerants of InS worms, but not AqS worms, were only sexual, indicating that sexual state regulation comprises two steps: (1) autonomous initiation of sexualizing substance production and (2) maintenance of the sexual state by continuous production of sexualizing substances. Next, InS neoblasts were characterized by transplantation, finding that they successfully engrafted, proliferated, and replaced all recipient cells. Under such conditions, the AS recipients of InS worm neoblasts, but not those of AqS worms, became sexual. These results clearly show that there is a neoblast-autonomous determination of reproductive strategy in planarians.

Annual rhythmicity in the switching of reproductive mode in planarians.

Species of planarians include both asexually reproducing individuals (reproduce through fission and regeneration) and sexually reproducing individuals (hermaphrodites that mate to produce cocoons). While some individuals can switch between the asexual and sexual modes of reproduction. In this study, we examined the reproductive modes and ploidy of Dugesia japonica and Dugesia ryukyuensis from three spring wells in Okinawa (Japan) during two consecutive years. D. japonica are mostly asexual and triploid. In contrast, only 40 % of D. ryukyuensis are asexual and triploid; the remaining are sexual, and diploid or triploid. The sexually reproductive season of D. ryukyuensis is winter. In July, the reproductive organs disappear, and the individuals start asexual reproduction through fission and regeneration. In January of the following year, the individuals develop ovaries and necessary reproductive organs and start sexual reproduction. When these species were lab-reared for a longer period, the reproductive cycles in three strains were repeated for three years. These results confirm that D. ryukyuensis population in Okinawa switches between reproductive modes on an annual cycle, even when kept under constant temperature and no light/dark cycle.

Morphological differences in tardigrade spermatozoa induce variation in gamete motility.

BACKGROUND: Fertilization is an event at the beginning of ontogeny. Successful fertilization depends on strategies for uniting female and male gametes that developed throughout evolutionary history. In some species of tardigrades, investigations of reproduction have revealed that released spermatozoa swim in the water to reach a female, after which the gametes are stored in her body. The morphology of the spermatozoa includes a coiled nucleus and a species-specific-length acrosome. Although the mating behaviour and morphology of tardigrades have been reported, the motility of male gametes remains unknown. Here, using a high-speed camera, we recorded the spermatozoon motilities of two tardigrades, Paramacrobiotus sp. and Macrobiotus shonaicus, which have longer and shorter spermatozoa, respectively. RESULTS: The movement of spermatozoa was faster in Paramacrobiotus sp. than in M. shonaicus, but the beat frequencies of the tails were equal, suggesting that the long tail improved acceleration. In both species, the head part consisting of a coiled nucleus and an acrosome did not swing, in contrast to the tail. The head part of Paramacrobiotus sp. spermatozoa swung harder during turning; in contrast, the tail of M. shonaicus moved more widely than the head. Finally, after mating, the spermatozoa that reached the female aggregated around the cloaca while waiting to enter her body in both tested species. CONCLUSIONS: This study provides results for the first observations and analyses of individual spermatozoon motility in tardigrades. A comparison of the spermatozoon movements of the two tardigrades suggested that the motilities of the male gametes were affected by morphological differences, where the longer spermatozoa swam faster and the shorter ones showed more stable swimming. Swimming was mainly induced by tail movement, but the long head of Paramacrobiotus sp. spermatozoa might be especially important for turning. In addition, observations of mated female cloacae suggested that the head parts of the spermatozoa were required for aggregation around the cloaca of a mated female.

Description of a model tardigrade Paramacrobiotus metropolitanus sp. nov. (Eutardigrada) from Japan with a summary of its life history, reproduction and genomics.

The genus Paramacrobiotus was erected in 2009 from the genus Macrobiotus, and 43 Paramacrobiotus species have been described to date. Although the first genome sequence in the genus was reported for the TYO strain of Paramacrobiotus sp., which is a dioecious species and has five bivalent chromosomes, its precise taxonomic identification remained undetermined. Here, we report its morphology, confirming the presence of a microplacoid, cuticular bulge on the inner side of legs IIII, and granulation on the inner side of legs IV under both light and electron microscopy, and smooth areoles on the egg shell, indicating that it differs from other described species. In addition, the previously described karyotype 2n=10 of this strain is clearly distinct from other species of the genus Paramacrobiotus, supporting the hypothesis that the strain represents a new species. Molecular analyses for the small and large ribosomal subunit (18S rDNA, 28S rDNA), the internal transcribed spacer 2 (ITS-2) and cytochrome C oxidase subunit I (COI) were also performed. The TYO strain is most similar in the analysed nuclear markers to Paramacrobiotus experimentalis Kaczmarek, Mioduchowska, Poprawa Roszkowska, 2020 and Paramacrobiotus sp. strain MG.002 (p-distances in 18S rDNA: 0.53%, 28S rDNA: 0.981.12%, and ITS-2: 9.9%), which corroborates with the overall morphological similarity between these taxa. Despite the close relationship between the TYO strain and P. experimentalis, the genetic species delimitation based on molecular analysis indicates that the TYO strain indeed is a distinct species. Therefore, this tardigrade is described here as Paramacrobiotus metropolitanus sp. nov.