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Mitosis in storage cells of the eutardigrade Richtersius coronifer

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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.

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... 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). ...
Chapter
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). ...
... 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. ...
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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. ...
Article
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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). ...
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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). ...
Article
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. ...
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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 ). ...
Chapter
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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.
Article
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].
Article
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.
Article
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.
Article
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.
Klassen und Ordungen des Tierreichs
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Acta Biologica Cracoviensa XXXVI
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Molting in Tardigrada. A review including new results on cuticle formation in Macrobiotus hufelandi
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Steroid hormones in Drosophila: how Ecdysone coordinates developmental signaling with cell growth and division
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