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Mitosis in storage cells of the eutardigrade Richtersius coronifer
Abstract
Although tardigrades are sometimes reported as eutelic animals, mitosis has been reported in several somatic tissues of adult eutardigrades. The occurrence of cell division in storage cells is particularly interesting in light of the important role that these cells play in the physiology of tardigrades. We present data on the occurrence of mitosis in storage cells of the eutardigrade Richtersius coronifer (Richters, 1903), and analyse mitotic cells in relation to different body characteristics, including egg development stage, moulting, gut content, body length, number and size of oocytes, and shape and size of the storage cells. Mitosis was present in ~20% of all animals, and was more frequent in juveniles than in adults. The proportion of cells with mitosis (‘mitotic index’) was low: 0.76% in juveniles and 1.47% in adults. In juveniles, none of the measured phenotypic characters had significant predictive power for mitosis, whereas in adult animals in moult or in late egg developmental or post-laying stage were more likely to have mitotic storage cells. The association with the later part of the moulting process was particularly strong. The low mitotic index and the strong association with moulting suggests that mitosis in storage cells may be connected with somatic growth rather than cell renewal, and that the purpose of cell division may relate to a need of more cells to support the enlarged body after moulting. However, the specific life cycle of tardigrades, where energy intake and depletion, egg development, and moulting is highly intertwined and synchronized, make conclusions about the functional role of mitosis in storage cells difficult, however, and more studies are needed to reveal the mechanisms inducing mitosis in these interesting cells.
... midgut, storage cells) they clearly vary in number. Other than the gonad (ovary, testis and ovotestis), in which mitoses and meioses frequently occur, mitoses have been frequently observed in storage cells (coelomocytes) and in the so-called transient cells in the beginning of the midgut (Bertolani 1970;Czernekova and Jönsson 2016). Increase in storage cell mitoses has been related to moulting and the late developmental stage of eggs (Czernekova and Jönsson 2016). ...
... Other than the gonad (ovary, testis and ovotestis), in which mitoses and meioses frequently occur, mitoses have been frequently observed in storage cells (coelomocytes) and in the so-called transient cells in the beginning of the midgut (Bertolani 1970;Czernekova and Jönsson 2016). Increase in storage cell mitoses has been related to moulting and the late developmental stage of eggs (Czernekova and Jönsson 2016). Variation in storage cell size and number has also been related to anhydrobiosis (Jönsson and Rebecchi 2002). ...
... Storage cells are also involved in vitellogenesis (Jönsson and Rebecchi 2002) and in the synthesis of vitellogenins (Hyra et al. 2016b). Mitoses frequently occur in storage cells (Bertolani 1970;Czernekova and Jönsson 2016). ...
Several cytological aspects have been considered in tardigrades. Firstly, the cell constancy which is not a true eutely being several mitoses present even after hatching, even though some organs, such epidermis and nervous ganglia, have the same cell number in juveniles and adults. The total number of these cells is species-specific. Then the ultrastructure of cuticle, epidermis, feeding and digestive apparatus, excretory and osmoregulatory organs, muscles, nerve cells, sensory cells and storage cells has been considered. Instead, the ultrastructure of the germ cells has been considered in the chapter on reproduction. With regard to chromosome number and shape, it has been observed that generally there is little difference among the species (n = 5 or n = 6), but several cases of polyploid populations exist, often very similar to diploid populations from a morphological point of view. In most cases the polyploid populations do not have males and reproduce by apomixis. Studies on the genome size have confirmed the presence of polyploid populations, as well as the presence of nuclei with multiple amounts of DNA within the same specimen. The genome size of the tardigrades is always relatively small and does not seem related to phylogenetic lineages. Studies on tardigrade genomes have placed this phylum at the centre of discussions on the evolution of Metazoa and have considered the role of horizontal gene transfer in animal evolution with contrasting results.
... Marcus (1929), citing a reduction in cells during late embryogenesis, similarly described the partial cell constancy in tardigrades as a secondary occurrence ("sekundäre Erscheinung") to differentiate it from true eutely, as is the case in nematodes (Sulston and Horvitz 1977). To date, mitotic cells have been observed in a number of different organs, although the total number of dividing cells per specimen is always low if present (Bertolani 1970a,b;Poprawa et al., 2015;Czerneková and Jönsson 2016). While these studies estimated the number of mitotic cells at any one time, it has never been shown how frequently or consistently cells divide in adult tardigrades. ...
... The frequency of cell divisions in whole tardigrades has been approximated previously based on the morphology of mitotic cells either in histological sections (Marcus 1929) or lacto-aceto-orcein-stained whole mounts (Bertolani 1970a,b;Czerneková and Jönsson 2016). All of these studies found low levels of cell divisions. ...
... All of these studies found low levels of cell divisions. Czerneková and Jönsson (2016) focused on storage cells, which have been hypothesized to have the highest rate of cell division of any cell type in tardigrades (Bertolani 1970a). Still, they estimated that less than 2% of storage cells are undergoing mitosis at any given point in time (Czerneková and Jönsson 2016). ...
... Females undergo DNA replication not only to produce oocytes through meiosis [23] but also to shed the cuticular exoskeleton during the last stage of the reproductive process (simplex stage) [23,87]. Mitotic cells are generally observed in the post-simplex stage [88]. Together, the regulation of DNA replication and meiosis is consistent with the production of mitotic cells and extensive replication of the epidermal layer [88,89]. ...
... Mitotic cells are generally observed in the post-simplex stage [88]. Together, the regulation of DNA replication and meiosis is consistent with the production of mitotic cells and extensive replication of the epidermal layer [88,89]. ...
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.
... The storage cells of tardigrades are cells of the body cavity that are involved primarily in nutritional maintenance [40,41] but also play a role in vitellogenesis [42,43] and possibly immune function [44]. These cells may therefore display variable internal morphologies and ultrastructure relating to their roles [41,45], a hypothesis that our data support based on the different internal gray values and textures seen in the storage cells in our CT scans. The sizes of storage cells have been measured in several studies in a number of different species [40,42,45,46]. ...
... These cells may therefore display variable internal morphologies and ultrastructure relating to their roles [41,45], a hypothesis that our data support based on the different internal gray values and textures seen in the storage cells in our CT scans. The sizes of storage cells have been measured in several studies in a number of different species [40,42,45,46]. Reuner et al. [40] performed the most comprehensive comparison (although H. exemplaris was not one of the species studied) and found that the number and size of storage cells varies greatly depending on the species. ...
Background:
Tardigrades (water bears) are microscopic invertebrates of which the anatomy has been well studied using traditional techniques, but a comprehensive three-dimensional reconstruction has never been performed. In order to close this gap, we employed X-ray computed tomography (CT), a technique that is becoming increasingly popular in zoology for producing high-resolution, three-dimensional (3D) scans of whole specimens. While CT has long been used to scan larger samples, its use in some microscopic animals can be problematic, as they are often too small for conventional CT yet too large for high-resolution, optics-based soft X-ray microscopy. This size gap continues to be narrowed with advancements in technology, with high-resolution imaging now being possible using both large synchrotron devices and, more recently, laboratory-based instruments.
Results:
Here we use a recently developed prototype lab-based nano-computed tomography device to image a 152 μm-long tardigrade at high resolution (200-270 nm pixel size). The resulting dataset allowed us to visualize the anatomy of the tardigrade in 3D and analyze the spatial relationships of the internal structures. Segmentation of the major structures of the body enabled the direct measurement of their respective volumes. Furthermore, we segmented every storage cell individually and quantified their volume distribution. We compare our measurements to those from published studies in which other techniques were used.
Conclusions:
The data presented herein demonstrate the utility of CT imaging as a powerful supplementary tool for studies of tardigrade anatomy, especially for quantitative volume measurements. This nanoCT study represents the smallest complete animal ever imaged using CT, and offers new 3D insights into the spatial relationships of the internal organs of water bears.
... The overall frequency of mitosis found in this study was similar to that reported in Czernekova and Jönsson [29], who found mitosis in 18.3% of the adult individuals of R. coronifer, and a mitotic index of 1.47% (based on individuals where mitotic cells were found). In that study it was also found that a higher frequency of mitosis was connected with the period of moulting which usually corresponds to the late phase of egg development. ...
... In that study it was also found that a higher frequency of mitosis was connected with the period of moulting which usually corresponds to the late phase of egg development. As mentioned above, that period is also characterized by smaller storage cells [15,26], but neither in the study by Czernekova and Jönsson [29] nor in the current study was cell size found to be associated with frequency of mitosis. Instead, the frequency of mitosis in storage cells tended to decline with the number of repeated desiccations and the total number of storage cells. ...
Tardigrades represent one of the main animal groups with anhydrobiotic capacity at any stage of their life cycle. The ability of tardigrades to survive repeated cycles of anhydrobiosis has rarely been studied but is of interest to understand the factors constraining anhydrobiotic survival. The main objective of this study was to investigate the patterns of survival of the eutardigrade Richtersius coronifer under repeated cycles of desiccation, and the potential effect of repeated desiccation on size, shape and number of storage cells. We also analyzed potential change in body size, gut content and frequency of mitotic storage cells. Specimens were kept under non-cultured conditions and desiccated under controlled relative humidity. After each desiccation cycle 10 specimens were selected for analysis of morphometric characteristics and mitosis. The study demonstrates that tardigrades may survive up to 6 repeated desiccations, with declining survival rates with increased number of desiccations. We found a significantly higher proportion of animals that were unable to contract properly into a tun stage during the desiccation process at the 5th and 6th desiccations. Also total number of storage cells declined at the 5th and 6th desiccations, while no effect on storage cell size was observed. The frequency of mitotic storage cells tended to decline with higher number of desiccation cycles. Our study shows that the number of consecutive cycles of anhydrobiosis that R. coronifer may undergo is limited, with increased inability for tun formation and energetic constraints as possible causal factors.
... Chile, Italy Ramazzotti, 1962;Maucci, 1973-74 M. cf. granulatum USA Jackson and Meyer, 2019 M. reticulatum Seychelles Pilato et al., 2002 M. katarzynae China, Colombia Kaczmarek et al., 2004;Caicedo et al., 2014;Melo et al., 2014;Londoño et al., 2015 M. krzysztofi Costa Rica, Peru, Colombia Kaczmarek and Michalczyk, 2007;Kaczmarek et al., 2014;Lisi et al., 2014;Melo et al., 2014; Horning et al., 1978 Chromosomal research on the Milnesium species from Japan There are several research studies on chromosomes in eutardigrades showing that the haploid numbers are mostly n = 5 or 6 (Rebecchi, 1991;Bertolani, 2001;Rebecchi et al., 2002Rebecchi et al., , 2003Czernekova and Jönsson, 2016;Guidetti et al., 2019;Sugiura et al., 2019). However, there has been only one report on the karyotype of Milnesium species, which indicated that the number was 2n = 10 in a German population of "Milnesium tardigradum" (Glätzer et al., 2005). ...
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.
... Chile, Italy Ramazzotti, 1962;Maucci, 1973-74 M. cf. granulatum USA Jackson and Meyer, 2019 M. reticulatum Seychelles Pilato et al., 2002 M. katarzynae China, Colombia Kaczmarek et al., 2004;Caicedo et al., 2014;Melo et al., 2014;Londoño et al., 2015 M. krzysztofi Costa Rica, Peru, Colombia Kaczmarek andMichalczyk, 2007;Kaczmarek et al., 2014;Lisi et al., 2014;Melo et al., 2014; Horning et al., 1978 Chromosomal research on the Milnesium species from Japan There are several research studies on chromosomes in eutardigrades showing that the haploid numbers are mostly n = 5 or 6 (Rebecchi, 1991;Bertolani, 2001;Rebecchi et al., 2002Rebecchi et al., , 2003Czernekova and Jönsson, 2016;Guidetti et al., 2019;Sugiura et al., 2019). However, there has been only one report on the karyotype of Milnesium species, which indicated that the number was 2n = 10 in a German population of "Milnesium tardigradum" (Glätzer et al., 2005). ...
The genetic diversity of the genus Ligidium in Hokkaido and Niigata, northern Japan, was investigated by analyzing the cytochrome c oxidase subunit 1 (CO1) region in the mitochondrial DNA (mtDNA). The genetic diversity in Hokkaido was much lower than that in Niigata. Nine different operational taxonomic units (OTUs) were identified. Only a single OTU, most likely Ligidium japonicum, was found in Hokkaido, whereas all nine OTUs were found in Niigata. Using the mtDNA evolutionary rate determined for the marine invertebrate Haptosquilla pulchella (Miers, 1880), population expansion for OTU1 in Hokkaido was estimated to have occurred at 12,600 years BP, suggesting that Ligidium underwent a bottleneck due to glacial cooling, and the population then expanded after postglacial warming. Assuming that the expansion of the OTU1 population occurred at 9600 years BP, when the sea surface temperature rose offshore of Tokachi in the Northwestern Pacific, the evolutionary rate (µ) of the mtDNA CO1 region in Ligidium is calculated as: 0.087 (95% confidence intervals: min: 0.042-max: 0.12) (substitutions/site/million years). The presence of a haplotype common to Hokkaido and Niigata implies that the haplotype migrated across the Tsugaru Strait. Considering that geological evidence indicates that the Tsugaru Strait was continuously present even during the last glacial maximum when the sea level was at its lowest, accidental transport by human beings or animals might have been critical to the migration of Ligidium.
... Notably, transcription-coupled-NER, plays an important role in removal of lesions from template DNA strands of actively transcribed genes [71]. The latter is in accordance with the observation that adult tardigrades seem to have low rates of mitotic cell divisions limited to specific cells and tissues [72,73], calling for a method of DNA repair that does not rely on replication. Our analyses indicated a conspicuous lack of members of the p53 family in the heterotardigrade E. cf. ...
Background:
Tardigrades are renowned for their ability to enter cryptobiosis (latent life) and endure extreme stress, including desiccation and freezing. Increased focus is on revealing molecular mechanisms underlying this tolerance. Here, we provide the first transcriptomes from the heterotardigrade Echiniscoides cf. sigismundi and the eutardigrade Richtersius cf. coronifer, and compare these with data from other tardigrades and six eukaryote models. Investigating 107 genes/gene families, our study provides a thorough analysis of tardigrade gene content with focus on stress tolerance.
Results:
E. cf. sigismundi, a strong cryptobiont, apparently lacks expression of a number of stress related genes. Most conspicuous is the lack of transcripts from genes involved in classical Non-Homologous End Joining. Our analyses suggest that post-cryptobiotic survival in tardigrades could rely on high fidelity transcription-coupled DNA repair. Tardigrades seem to lack many peroxins, but they all have a comprehensive number of genes encoding proteins involved in antioxidant defense. The "tardigrade unique proteins" (CAHS, SAHS, MAHS, RvLEAM), seem to be missing in the heterotardigrade lineage, revealing that cryptobiosis in general cannot be attributed solely to these proteins. Our investigation further reveals a unique and highly expressed cold shock domain. We hypothesize that the cold shock protein acts as a RNA-chaperone involved in regulation of translation following freezing.
Conclusions:
Our results show common gene family contractions and expansions within stress related gene pathways in tardigrades, but also indicate that evolutionary lineages have a high degree of divergence. Different taxa and lineages may exhibit unique physiological adaptations towards stress conditions involving possible unknown functional homologues and/or novel physiological and biochemical mechanisms. To further substantiate the current results genome assemblies coupled with transcriptome data and experimental investigations are needed from tardigrades belonging to different evolutionary lineages.
... Their numbers seem to decline during starvation and thus vary with the nutritional state of the animal (Weglarska 1975). Accordingly, it has been shown that mitosis occurs in storage cells (as well as in other cell types; Bertolani 1970a, b;Czerneková and Jönsson 2016;Hyra et al. 2016b), which strongly questions whether tardigrades are truly eutelic as sometimes reported. Interestingly, storage cells may additionally play a defensive role, phagocytizing bacteria ). ...
Tardigrades are microscopic aquatic bilaterians that despite their size have a complex morphology and anatomy. These minute animals need a surrounding film of water to be active, and tardigrades residing in terrestrial environments live in moist microhabitats. The latter species are referred to as semiterrestrial, and they endure rapid dehydration by losing water over highly permeable integuments while forming a quiescent tun. Species of the class Eutardigrada are predominantly semiterrestrial and limnic with a relatively uniform morphology and anatomy. Heterotardigrades (class Heterotardigrada) comprising echiniscoideans and arthrotardigrades are much more diverse with highly variable external morphologies, ranging from clearly segmented species, over species with extremely large external sense organs and cuticular wings to species with a more “bear”-like eutardigrade body outline. In this chapter, we review the current understanding of tardigrade morphology and functional anatomy and put focus on tardigrade diversity emphasizing the often neglected marine arthrotardigrades. We give an introduction to the structure and function of major tardigrade organ systems, including the integument, body cavity, and digestive, muscular, nervous, and reproductive systems, and we highlight the diversity of tardigrade sensory appendages and overall external morphology.
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 shows high levels of diversity, factors such as the Doublesex-Mab-3-related transcription factor (DMRT) are highly conserved throughout animals. Species of the phylum Tardigrada exhibits remarkable diversity in morphology and behavior between sexes, suggesting a pathway regulating such dimorphism. Despite the wealth of genomic and zoological knowledge accumulated in recent studies, the sexual differences in tardigrades genomes have not been identified. In this 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 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 lacks the doublesex ortholog, suggesting doublesex emerged after the divergence of Tardigrada. In contrast to sex-specific expression, no evidence of genomic difference between the sexes were found. We also identified several anhydrobiosis genes exhibiting sex-biased expression, possibly suggesting a 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 would provide 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.
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.
Richtersius coronifer, the nominal species for the family Richtersiidae and a popular laboratory model, exemplifies a common problem in modern tardigrade taxonomy. Despite undeniable progress in the field, many old and incomplete descriptions of taxa hinder both species delimitation and the estimation of species diversity and distribution. Although for over a century this species has been recorded throughout the world, recent research indicates that records to date are likely to represent a species complex rather than a single cosmopolitan species. However, in order to recognise and name species diversity within the complex, an integrative redescription of the nominal species is first needed. Here, we describe an R. coronifer population collected from Spitsbergen, i.e., one of the two localities mentioned in the original description, with detailed morphological and morphometric data associated with standard DNA sequences of four standard genetic markers (18S rRNA, 28S rRNA, ITS-2, and COI) and supported by transcriptome sequencing. We propose replacement of the neotype designated in 1981 by Maucci and Ramazzotti, as it is impossible to verify whether the existing neotype is conspecific with specimens studied by Richters in 1903 and 1904. Finally, using newly obtained cytochrome c oxidase subunit I (COI) sequences of populations from Spitsbergen, Italy, Poland, and Greece together with sequences deposited in GenBank (China, Greenland, Italy, Mongolia), we performed genetic species delimitation, which indicated seven distinct potential species within the genus Richtersius, in addition to the nominal taxon. This study marks a starting point for further research on the taxonomy of and species diversity within the genus. Moreover, this work has the potential to be the first tardigrade redescription to provide both genetic barcodes and a transcriptome of the species in question.
Important contributions have been made to the systematics of Eutardigrada in recent years, but these have also revealed that several taxa are polyphyletic and that cryptic species are present. To shed light on the taxonomy and systematic position of the genus Richtersius (Eutardigrada, Macrobiotoidea), six populations attributed to Richtersius coronifer were collected and analysed from morphological (light and scanning electron microscopy) and molecular (mitochondrial cytochrome oxidase subunit 1, 18S, 28S) points of view. In particular, a new morphometric index (claw common tract: length of the common tract of the claw/total claw length × 100) and a new morphological character (stalk system) were introduced. Our integrative study was able to unveil the ‘cryptic’ species diversity within Richtersius, showing that the genus contains more than one evolutionary lineage. A morphological peculiarity in the animals of all lineages is the dimorphism in the morphology of the cuticle. Cuticular pores are present in the newborns and are lost with the first moult; this morphological change represents a novelty in the life cycle of eutardigrades. The phylogenetic analyses carried out on Richtersius populations and other Macrobiotoidea show that Richtersius is closely related to Macrobiotus islandicus, whereas Adorybiotus granulatus is more related to Richtersius and M. islandicus than to other members of the genus Macrobiotus (type genus of Macrobiotidae); therefore, the genus Macrobiotus and the family Macrobiotidae are not monophyletic. Based on these results, the new genus Diaforobiotus (for M. islandicus) and the new family Richtersiidae (composed of Richtersius, Diaforobiotus gen. nov., and Adorybiotus) are established.
Three populations of Macrobiotus pseudohufelandi (Eutardigrada, Macrobiotidae) were studied with karyological techniques not previously utilized for tardigrades. These observations provided detailed information on the karyology of these animals, particularly single chromosome morphology. A diploid amphimictic cytotype and a triploid thelytokous parthenogenetic cytotype were confirmed. In addition, a new tetraploid thelytokous parthenogenetic cytotype was found. Tetraploidy, as defined by chromosome number, was confirmed by DNA-Feulgen content evaluations. Indeed, the minimum DNA content in the tetraploid population is about four fold that in sperm from the diploid population.
Tardigrades are known as one of the most radiation tolerant animals on Earth, and several studies on tolerance in adult tardigrades have been published. In contrast, very few studies on radiation tolerance of embryonic stages have been reported. Here we report a study on tolerance to gamma irradiation in eggs of the eutardigrade Richtersius coronifer. Irradiation of eggs collected directly from a natural substrate (moss) showed a clear dose-response, with a steep decline in hatchability at doses up to 0.4 kGy followed by a relatively constant hatchability around 25% up to 2 kGy, and a decline to ca. 5% at 4 kGy above which no eggs hatched. Analysis of the time required for eggs to hatch after irradiation (residual development time) showed that hatching of eggs after exposure to high doses of gamma radiation was associated with short residual development time. Since short residual development time means that the egg was irradiated at a late developmental stage, this suggests that eggs were more tolerant to radiation late in development. This was also confirmed in another experiment in which stage of development at irradiation was controlled. No eggs irradiated at the early developmental stage hatched, and only one egg at middle stage hatched, while eggs irradiated in the late stage hatched at a rate indistinguishable from controls. This suggests that the eggs are more sensitive to radiation in the early stages of development, or that tolerance to radiation is acquired only late in development, shortly before the eggs hatch, hypotheses that are not mutually exclusive. Our study emphasizes the importance of considering specific cell cycle phases and developmental stages in studies of tolerance to radiation in tardigrades, and the potential importance of embryonic studies in revealing the mechanisms behind the radiation tolerance of tardigrades and other cryptobiotic animals.
All metazoans are exposed to a wide range of microbes and have evolved complex immune defenses used to repel infectious agents. Coelomocytes play a key role in the defense reactions of most invertebrates. They are involved in important immune functions, such as phagocytosis, encapsulation, graft rejection, and inflammation, as well as the synthesis and secretion of several humoral factors especially in annelids and echinoderms. Coelomocytes in nematodes are variable in shapes from round, ovoid, cuboidal, and spindle-shaped to stellate or branched cells that are found usually at fixed positions in the pseudocoelom. Their number usually varies from 2 to 6. The model nematode, C. elegans lacks an adaptive immune system and the coelomocytes are capable of endocytosis, but their involvement in phagocytosis of bacteria seems unlikely. The aim of this review is to evaluate current knowledge on coelomocytes of invertebrates with special reference to nematodes. The morphology and structure of these coelomocytes are discussed along with their origin. Their relative positions and diversity in different nematode groups have also been discussed and illustrated.
Tardigrades are known to survive high doses of ionizing radiation. However, there have been no reports about radiation effects in tardigrades under culture conditions. In this study, we investigated tolerance of the tardigrade, Milnesium tardigradum, against gamma-rays and heavy ions by determining short-term or long-term survival, and reproductive ability after irradiation.
Hydrated and anhydrobiotic animals were exposed to gamma-rays (1000 - 7000 Gy) or heavy ions (1000 - 8000 Gy) to evaluate short-term survival at 2, 24 and 48 h post-irradiation. Long-term survival and reproduction were observed up to 31 days after irradiation with gamma-rays (1000 - 4000 Gy).
At 48 h after irradiation, median lethal doses were 5000 Gy (gamma-rays) and 6200 Gy (heavy ions) in hydrated animals, and 4400 Gy (gamma-rays) and 5200 Gy (heavy ions) in anhydrobiotic ones. Gamma-irradiation shortened average life span in a dose-dependent manner both in hydrated and anhydrobiotic groups. No irradiated animals laid eggs with one exception in which a hydrated animal irradiated with 2000 Gy of gamma-rays laid 3 eggs, and those eggs failed to hatch, whereas eggs produced by non-irradiated animals hatched successfully.
M. tardigradum survives high doses of ionizing radiation in both hydrated and anhydrobiotic states, but irradiation with >1000 Gy makes them sterile.
The impact of starvation and anhydrobiosis on the number and size of the storage cells in the tardigrade species Milnesium tardigradum, Paramacrobiotus tonollii and Macrobiotus sapiens was investigated to gain more insight on the energetic side of anhydrobiosis. Storage cells are free floating cells within the body cavity of tardigrades and are presumed to store and release energy in form of glycogen, protein and fat to maintain a constant nutrient regime for the other tissues. The body size of the animals was not correlated with the size of the storage cells, however, M. tardigradum the largest species analysed also had the largest storage cells. A reduction in the size of the storage cells is apparent in all three species after seven days of starvation. A seven-day period of anhydrobiosis leads to a decrease in cell size in M. tardigradum but not in P. tonollii and M. sapiens. Although M. sapiens was raised on green algae, and M. tardigradum and P. tonollii were fed with rotifers and nematodes this difference in nourishment was not reflected in the response of the storage cells to anhydrobiosis.
The Author underlines the utility of pointing out the per cent ratio between the length of any measurable strucutre and the length of the buccal tube (per cent ratio indicated with the abbreviation pt). The Author demonstrates that the distance between the insertion point of the stylet supports and the anterior edge of the stylet sheaths is a good specific character: the per cent ratio between such a distance and the buccal tube length shows a very little intraspecific variability and it does not appear correlated either with the body length or the length of the buccal tube.
We studied the life history of tardigrades with a particular focus on the maturative patterns of the ovary and testis. Specimens collected in nature belonging to four species of one freshwater and two semiterrestrial genera of eutardigrades were examined. The females of all examined species are always iteroparous; they have several maturative cycles with synchronously developing oocytes. Four maturative stages can be distinguished in each ovarian cycle, which is clearly correlated with moulting. In contrast, gametocyte maturation does not seem to be correlated with moulting in males and varies markedly in the considered genera. The testes of adult specimens of Macrobiotus always contain both mature spermatozoa and cells at earlier stages in spermatogenesis (continuous maturation). The testis of Pseudobiotus megalonyx, the only freshwater species examined, shows a gradual increase in spermatozoa that in the end completely fill the gonad (progressive maturation, tied to a semelparous life cycle). The testis in adult males of Amphibolus is either filled exclusively with spermatozoa or alternatively with immature germinal cells, revealing an iteroparous condition similar to that of the females (cyclical maturation). The different types of gonad maturation in males seem to be correlated with habitat and the capacity to enter cryptobiosis.
Allozymes were assessed by starch gel electrophoresis in 3 populations of a eutardigrade, Richtersius coronifer, with different reproductive modes. One population from Italy (with 2 sub-populations) was amphimictic and 2 populations (1 from Italy and 1 from Sweden) were parthenogenetic. All populations, irrespective of their reproductive mode, were diploid with the same chromosome number (2n=12) and had bivalents in the oocytes. Of the 14 loci analyzed, only 3 were polymorphic. The amphimictic population had a higher degree of genetic variability (mean heterozygosity >0.25) than the parthenogenetic populations (mean heterozygosity of the 2 populations <0.01). In all female populations, allele frequencies at all 3 loci deviated from Hardy-Weinberg equilibria due to heterozygote deficiency. These results support a hypothesis of automictic parthenogenesis in R. coronifer.
Changes in the trehalose level in the tardigrade Adorybiotus coronifer were observed during induction of, and arousal from, anhydrobiosis. A trehalose accumulation surpassing 1.6% dry weight (d.w.) in anhydrobiotic animals collected dry on their biotope was rapidly reduced on rehydration, reaching the level of active animals (0.1–0.2% d.w.) after 6 hours. Tardigrades dried while embedded in sand in the laboratory accumulated trehalose from 0.1% to 2.3% d.w. within 5–7 hours.
Induction of aerobic acidosis in arousing tardigrades by CO2 perfusion reversibly arrested reactivation for at least 36 hours and induced a reduction in anabolic and catabolic activities, measured as a significant reduction in trehalose degradation, and a sevenfold reduction in the rate of protein synthesis.
These data support the hypotheis that trehalose generally serves a protective role in desiccationtolerant Metazoa, but indicate that tardigrades require only a moderate level for efficient protection.
We report a study on anhydrobiotic survival in the eutardigrades Richtersius coronifer and Ramazzottius oberhaeuseri. In each of these species, we investigated the anhydrobiotic survival of two populations, one from Sweden and one from Italy. We found that anhydrobiotic survival was similar in the Swedish and the Italian populations in both species, indicating no divergence with respect to anhydrobiotic capacity. Body size had a strong effect on the probability to recover from anhydrobiosis, but the effect was in opposite direction in the two tardigrade species. Ramazzottius oberhaeuseri had a considerable higher overall survival (66%) than R. coronifer (40%). This result is in line with earlier studies that found R. oberhaeuseri to have a very high water-retentive capacity.
The impact of starvation and anhydrobiosis on the number and size of the storage cells in the tardigrade species Milnesium tardigradum, Paramacrobiotus tonollii and Macrobiotus sapiens was investigated to gain more insight on the energetic side of anhydrobiosis. Storage cells are free floating cells within the body cavity of tardigrades and are presumed to store and release energy in form of glycogen, protein and fat to maintain a constant nutrient regime for the other tissues. The body size of the animals was not correlated with the size of the storage cells, however, M. tardigradum the largest species analysed also had the largest storage cells. A reduction in the size of the storage cells is apparent in all three species after seven days of starvation. A seven-day period of anhydrobiosis leads to a decrease in cell size in M. tardigradum but not in P. tonollii and M. sapiens. Although M. sapiens was raised on green algae, and M. tardigradum and P. tonollii were fed with rotifers and nematodes this difference in nourishment was not reflected in the response of the storage cells to anhydrobiosis [Current Zoology 56 (2): 259–263, 2010].
The ability of some animal taxa (e.g., nematodes, rotifers, and tardigrades) to enter an ametabolic (cryptobiotic) state is well known. Nevertheless, the phenotypic factors affecting successful anhydrobiosis have rarely been investigated. We report a laboratory study on the effects of body size, reproductive condition, and energetic condition on anhydrobiotic survival in a population of the eutardigrade Richtersius coronifer. Body size and energetic condition interacted in affecting the probability of survival, while reproductive condition had no effect. Large tardigrades had a lower probability of survival than medium-sized tardigrades and showed a positive response in survival to energetic condition. This suggests that energy constrained the possibility for large tardigrades to enter and to leave anhydrobiosis. As a possible alternative explanation for low survival in the largest specimens we discuss the expression of senescence. In line with the view that processes related to anhydrobiosis are connected with energetic costs we documented a decrease in the size of storage cells over a period of anhydrobiosis, showing for the first time that energy is consumed in the process of anhydrobiosis in tardigrades.
Tardigrades have a reputation of being extremely tolerant to extreme environmental conditions including tolerance to ionizing radiation while in a desiccated, anhydrobiotic state. However, the evidence for radio-tolerance in tardigrades is based on only one previous report, and there is an obvious need for complementary studies. In this paper we report an investigation on radio-tolerance in desiccated and hydrated specimens of the eutardigrade Richtersius coronifer.
Groups of 30 - 50 tardigrades were exposed to gamma-radiation at doses between 1.0 - 9.0 (anhydrobiotic animals) or 0.5 - 5.0 (hydrated animals) kGy and the animals were followed until all were dead. Radiation tolerance of both desiccated and hydrated tardigrades was studied.
Both desiccated and hydrated animals irradiated with 0.5 and 1 kGy did not deviate in survival from the control groups. Animals from all exposed groups underwent their moulting and egg production cycle, but at decreasing frequency for doses above 1 kGy. No eggs laid by irradiated animals hatched, while eggs laid by controls did so.
Our study suggests that radiation tolerance in tardigrades is not due to biochemical protectants connected with the desiccated state. Rather, cryptobiotic tardigrades may rely on efficient mechanisms of DNA repair, the nature of which is currently unknown.
Tyrosinase activity has been demonstrated ultra-structurally in the cuticle of the eutardigrade Macrobiotus hufelandi. The enzyme could be localized in the outer layer (=epicuticle) of the integument. A weak deposition of reaction product was also seen in the cytoplasm of storage cells, free floating in the haemolymph.
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