All for one: changes in mitochondrial morphology and activity during syncytial oogenesis†.

The syncytial groups of germ cells (germ-line cysts) forming in ovaries of clitellate annelids are an attractive model to study mitochondrial stage-specific changes. Using transmission electron microscopy, serial block-face scanning electron microscopy, and fluorescent microscopy, we analyzed the mitochondria distribution and morphology and the state of membrane potential in female cysts in Enchytraeus albidus. We visualized in 3D at the ultrastructural level mitochondria in cysts at successive stages: 2-celled, 4-celled, 16-celled cysts, and cyst in advanced oogenesis. We found that mitochondria form extensive aggregates-they are fused and connected into large and branched mitochondrial networks. The most extensive networks are formed with up to 10 000 fused mitochondria, whereas individual organelles represent up to 2% of the total mitochondrial volume. We classify such a morphology of mitochondria as a dynamic hyperfusion state and suggest that this can maintain their high activity and intensify the process of cellular respiration within the syncytial cysts. We found some individual mitochondria undergoing degradation, which implies that damaged mitochondria are removed from networks for their final elimination. As growing oocytes were shown to possess less active mitochondria than the nurse cells, the high activity of mitochondria in the nurse cells and their dynamic hyperfusion state are attributed to serve the needs of the growing oocyte. In addition, we measured by calorimetry the total antioxidant capacity of germ-line cysts in comparison with somatic tissue, and it suggests that antioxidative defense systems, together with mitochondrial networks, can effectively protect germ-line mitochondria from damage.

3,4-Dehydro-L-proline Induces Programmed Cell Death in the Roots of Brachypodium distachyon.

As cell wall proteins, the hydroxyproline-rich glycoproteins (HRGPs) take part in plant growth and various developmental processes. To fulfil their functions, HRGPs, extensins (EXTs) in particular, undergo the hydroxylation of proline by the prolyl-4-hydroxylases. The activity of these enzymes can be inhibited with 3,4-dehydro-L-proline (3,4-DHP), which enables its application to reveal the functions of the HRGPs. Thus, to study the involvement of HRGPs in the development of root hairs and roots, we treated seedlings of Brachypodium distachyon with 250 µM, 500 µM, and 750 µM of 3,4-DHP. The histological observations showed that the root epidermis cells and the cortex cells beneath them ruptured. The immunostaining experiments using the JIM20 antibody, which recognizes the EXT epitopes, demonstrated the higher abundance of this epitope in the control compared to the treated samples. The transmission electron microscopy analyses revealed morphological and ultrastructural features that are typical for the vacuolar-type of cell death. Using the TUNEL test (terminal deoxynucleotidyl transferase dUTP nick end labelling), we showed an increase in the number of nuclei with damaged DNA in the roots that had been treated with 3,4-DHP compared to the control. Finally, an analysis of two metacaspases' gene activity revealed an increase in their expression in the treated roots. Altogether, our results show that inhibiting the prolyl-4-hydroxylases with 3,4-DHP results in a vacuolar-type of cell death in roots, thereby highlighting the important role of HRGPs in root hair development and root growth.

Can Ceylon Leadwort (Plumbago zeylanica L.) Acclimate to Lead Toxicity?-Studies of Photosynthetic Apparatus Efficiency.

Ceylon leadwort (Plumbago zeylanica) is ornamental plant known for its pharmacological properties arising from the abundant production of various secondary metabolites. It often grows in lead polluted areas. The aim of presented study was to evaluate the survival strategy of P. zeylanica to lead toxicity via photosynthetic apparatus acclimatization. Shoots of P. zeylanica were cultivated on media with different Pb concentrations (0.0, 0.05, and 0.1 g Pb∙l-1). After a four-week culture, the efficiency of the photosynthetic apparatus of plants was evaluated by Chl a fluorescence measurement, photosynthetic pigment, and Lhcb1, PsbA, PsbO, and RuBisCo protein accumulation, antioxidant enzymes activity, and chloroplast ultrastructure observation. Plants from lower Pb concentration revealed no changes in photosynthetic pigments content and light-harvesting complex (LHCII) size, as well as no limitation on the donor side of Photosystem II Reaction Centre (PSII RC). However, the activity and content of antioxidant enzymes indicated a high risk of limitation on the acceptor side of Photosystem I. In turn, plants from 0.1 g Pb∙l-1 showed a significant decrease in pigments content, LHCII size, the amount of active PSII RC, oxygen-evolving complex activity, and significant remodeling of chloroplast ultrastructure indicated limitation of PSII RC donor side. Obtained results indicate that P. zeylanica plants acclimate to lead toxicity by Pb accumulation in roots and, depending on Pb concentration, by adjusting their photosynthetic apparatus via the activation of alternative (cyclic and pseudocyclic) electron transport pathways.

Ovaries of the white worm (Enchytraeus albidus, Annelida, Clitellata) are composed of 16-celled meroistic germ-line cysts.

The paired ovaries of E. albidus are like a bunch of grapes and are composed of clearly separated units, syncytial germ cysts (clusters), which are surrounded by a thin layer of somatic cells. Each cyst maintains the connection with the ovary by an extended stalk that is composed of somatic cells. The spatial architecture of the germ-line cysts found in E. albidus is the same as in other clitellate annelids that have been studied to date. As a rule, germ cells are located at the cyst periphery and each has only one ring canal that connects it to the common and centrally located cytoplasmic mass, the cytophore. Here we present data about the F-actin and microtubular cytoskeleton and some molecular components of the germ-line cysts. We show that the ring canals have an inner rim that is enriched with microfilaments and proteins that contain phosphotyrosine. The microtubules form a loose network in the cytoplasm of the oocyte and nurse cells; moreover, some of them pass through the ring canals to the cytophore. Numerous microtubules are also located in the somatic cells. The germ-line cysts in E. albidus ovaries consist of 16 cells, which is the lowest known number of interconnected germ cells within clitellate annelids. During oogenesis, the fate of interconnected germ cells differentiates and only one cell develops as the future egg, while the other 15 become nurse cells. This differentiation means ovary meroism. The nurse cells gather cell organelles and storage material that then pass through the ring canals and cytophore moving towards the growing oocyte. At the end of oogenesis, the vitellogenic oocyte surrounds the siblings' cells together with the cytophore and engulfs their remnants into the ooplasm. No morphological or molecular markers of the apoptosis of the nurse cells were found. Moreover, the nurse cells did not undergo polyploidisation. The measured DNA level was 4C, which indicates that these cells are not highly-specialised.

Organ and Tissue-Specific Localisation of Selected Cell Wall Epitopes in the Zygotic Embryo of Brachypodium distachyon.

The plant cell wall shows a great diversity regarding its chemical composition, which may vary significantly even during different developmental stages. In this study, we analysed the distribution of several cell wall epitopes in embryos of Brachypodium distachyon (Brachypodium). We also described the variations in the nucleus shape and the number of nucleoli that occurred in some embryo cells. The use of transmission electron microscopy, and histological and immunolocalisation techniques permitted the distribution of selected arabinogalactan proteins, extensins, pectins, and hemicelluloses on the embryo surface, internal cell compartments, and in the context of the cell wall ultrastructure to be demonstrated. We revealed that the majority of arabinogalactan proteins and extensins were distributed on the cell surface and that pectins were the main component of the seed coat and other parts, such as the mesocotyl cell walls and the radicula. Hemicelluloses were localised in the cell wall and outside of the radicula protodermis, respectively. The specific arrangement of those components may indicate their significance during embryo development and seed germination, thus suggesting the importance of their protective functions. Despite the differences in the cell wall composition, we found that some of the antibodies can be used as markers to identify specific cells and the parts of the developing Brachypodium embryo.

5-Azacitidine Induces Cell Death in a Tissue Culture of Brachypodium distachyon.

Morphological and histological observations revealed that, at a concentration of 50 µM, 5-azacitidine (5-azaC) totally inhibited the induction of embryogenic masses (EM), while the cultivation of explants (zygotic embryos; ZEs) in the presence of 5 µM of 5-azaC led to the formation of a callus with EM in 10% of the cases. Transmission electron microscopy (TEM) analyzes revealed the presence of the morphological and ultrastructural features that are typical for the vacuolar type of cell death in the callus cells that were treated. A TUNEL assay confirmed the presence of DNA double-strand breaks for the callus cells that had been treated with both 5 and 50 µM 5-azaC concentrations. Analysis of the gene expression of selected cell death markers demonstrated a reduced expression of metacaspase, protein executer 1 (EX1), and thioredoxin (TRX) in the callus cells that had been treated compared to the control culture. The strongest increase in the gene activity was characteristic for glutathione S-transferase (GST). Our studies also included an analysis of the distribution of some arabinogalactan proteins (AGPs) and extensin epitopes, which can be used as markers of cells that are undergoing death in a Brachypodium distachyon tissue culture.

Influence of the modifiers in polyol method on magnetically induced hyperthermia and biocompatibility of ultrafine magnetite nanoparticles.

Magnetite nanoparticles (Fe3O4 NPs) are widely tested in various biomedical applications, including magnetically induced hyperthermia. In this study, the influence of the modifiers, i.e., urotropine, polyethylene glycol, and NH4HCO3, on the size, morphology, magnetically induced hyperthermia effect, and biocompatibility were tested for Fe3O4 NPs synthesized by polyol method. The nanoparticles were characterized by a spherical shape and similar size of around 10 nm. At the same time, their surface is functionalized by triethylene glycol or polyethylene glycol, depending on the modifiers. The Fe3O4 NPs synthesized in the presence of urotropine had the highest colloidal stability related to the high positive value of zeta potential (26.03 ± 0.55 mV) but were characterized by the lowest specific absorption rate (SAR) and intrinsic loss power (ILP). The highest potential in the hyperthermia applications have NPs synthesized using NH4HCO3, for which SAR and ILP were equal to 69.6 ± 5.2 W/g and 0.613 ± 0.051 nHm2/kg, respectively. Their application possibility was confirmed for a wide range of magnetic fields and by cytotoxicity tests. The absence of differences in toxicity to dermal fibroblasts between all studied NPs was confirmed. Additionally, no significant changes in the ultrastructure of fibroblast cells were observed apart from the gradual increase in the number of autophagous structures.

A microscopy investigation of the complex problem of infertility of insect hybrids. Studies on the reproductive systems, eggshells, and karyotypes of the representatives of the genus Platymeris (Heteroptera, Reduviidae) and their hybrids.

Hybridisation is still a widely studied phenomenon that allows us to look at some processes differently. However, obtaining fertile hybrids, which we might consider in the long term as precursors of a new species, is still a field that requires research. Much of the research done so far indicates that hybrids are sterile - either sex or both. It is influenced by various mechanisms, both prezygotic and postzygotic reproductive isolation. Thanks to the use of light and transmission electron microscopy, our research has made it possible to understand, at least partially, the causes of infertility in male hybrids resulting from the crossing of two Afrotropical species of the genus Platymeris Laporte, 1833. The analysis of microscopic images showed that one of the possible causes of the infertility of hybrids might be spermatogenesis, during which sperm cells were not formed. In turn, the use of scanning electron microscopy revealed potential abnormalities in the structure of the hybrid eggshell. Moreover, karyotyping analyses suggest possible causes of infertility at the genetic level.

Ultra-Morphology and Mechanical Function of the Trichoideum Sensillum in Nabis rugosus (Linnaeus, 1758) (Insecta: Heteroptera: Cimicomorpha).

The present study aims to investigate the morphological features of the antennal sensilla by using SEM and TEM. The construction of a 3D model of trichoideum sensillum using Amira software is presented in this paper. Five sensillum types, namely trichoideum, chaeticum, campaniformium, coeloconicum, and basiconicum, were recorded. This model exhibits the mechanosensillum components, including the embedded hair in a socket attached by the joint membrane and the dendrite connected to the hair base passing through the cuticle layers. TEM images present the dendrite way, micro-tubules inside the dendritic sheath, and terminal structure of the tubular dendrite body and so-called companion cells included in the receptor, e.g., tormogen and trichogen. The parameters noted for the external structure and ultrastructure of the mechano-receptor indicate that they are specific to a particular type of sensillum and would be useful in developing the model for a biosensor. Results show that bio-inspired sensors can be developed based on morphological and ultrastructural studies and to conduct mechanical studies on their components.

Ovaries and testes of Lithobius forficatus (Myriapoda, Chilopoda) react differently to the presence of cadmium in the environment.

Proper reproduction depends on properly functioning gonads (ovaries and testes). Many xenobiotics, including heavy metals, can cause changes in somatic and germ line cells, thus damaging the reproductive capacity. The aim of this study was to investigate the effect of the heavy metal cadmium on the gonads, including germ line and somatic cells. It is important to determine whether cell death processes are triggered in both types of cells in the gonads, and which gonads are more sensitive to the presence of cadmium in the environment. The research was conducted on the soil-dwelling arthropod Lithobius forficatus (Myriapoda, Chilopoda), which is common for European fauna. Animals were cultured in soil supplemented with Cd for different periods (short- and long-term treatment). Gonads were isolated and prepared for qualitative and quantitative analysis, which enabled us to describe all changes which appeared after both the short- and long-term cadmium treatment. The results of our study showed that cadmium affects the structure and ultrastructure of both gonads in soil-dwelling organisms including the activation of cell death processes. However, the male germ line cells are more sensitive to cadmium than female germ line cells. We also observed that germ line cells are protected by the somatic cells of both gonads.

Hazards related to the presence of cadmium in food - Studies on the European soil centipede, Lithobius forficatus.

The soil is an environment rich in numerous potentially toxic substances/elements when present at elevated concentrations. They can be transported through the successive levels of the trophic chain. Animals living in a contaminated environment or eating contaminated food can accumulate potentially toxic elements in their bodies. One of the potentially toxic metals is cadmium, which accumulates significantly in soils. The aim of our research was to evaluate the changes caused by cadmium supplied with the food administered to invertebrates living in uncontaminated soil. The results were compared with those obtained for animals raised in contaminated soil, where cadmium entered the body via the epidermis. As the material for studies, we chose a common European soil centipede, Lithobius forficatus. Adult specimens were divided into the following experimental groups: C - control animals, Cd12 and Cd45 - animals fed with Chironomus larvae maintained in water containing 80 mg/l CdCl2, for 12 and 45 days, respectively. The material was analyzed using qualitative and quantitative analysis (transmission electron microscopy, confocal microscopy, flow cytometry, atomic absorption spectrometry). Eventually, we can conclude that the digestive system is an effective barrier against the effects of toxic metals on the entire organism, but among the gonads, ovaries are more protected than testes, however, this protection is not sufficient. Accumulation of spherites and mitochondrial alterations are probably involved in survival mechanisms of tissues after Cd intoxication.

Verification of Hypsibius exemplaris Gasiorek et al., 2018 (Eutardigrada; Hypsibiidae) application in anhydrobiosis research.

Anhydrobiosis is considered to be an adaptation of important applicative implications because it enables resistance to the lack of water. The phenomenon is still not well understood at molecular level. Thus, a good model invertebrate species for the research is required. The best known anhydrobiotic invertebrates are tardigrades (Tardigrada), considered to be toughest animals in the world. Hypsibius. exemplaris is one of the best studied tardigrade species, with its name "exemplaris" referring to the widespread use of the species as a laboratory model for various types of research. However, available data suggest that anhydrobiotic capability of the species may be overestimated. Therefore, we determined anhydrobiosis survival by Hys. exemplaris specimens using three different anhydrobiosis protocols. We also checked ultrastructure of storage cells within formed dormant structures (tuns) that has not been studied yet for Hys. exemplaris. These cells are known to support energetic requirements of anhydrobiosis. The obtained results indicate that Hys. exemplaris appears not to be a good model species for anhydrobiosis research.

Star Polymers as Non-Viral Carriers for Apoptosis Induction.

Apoptosis is a widely controlled, programmed cell death, defects in which are the source of various diseases such as neurodegenerative diseases as well as cancer. The use of apoptosis in the therapy of various human diseases is of increasing interest, and the analysis of the factors involved in its regulation is valuable in designing specific carriers capable of targeting cell death. Highly efficient and precisely controlled delivery of genetic material by low-toxic carriers is one of the most important challenges of apoptosis-based gene therapy. In this work, we investigate the effect of the star polymer with 28 poly(N,N'-dimethylaminoethyl methacrylate) arms (STAR) on human cells, according to its concentration and structure. We show that star polymer cytotoxicity increases within its concentration and time of cells treatment. Except for cytotoxic effect, we observe morphological changes such as a shrinkage, loss of shape and begin to detach. We also prove DNA condensation after star polymer treatment, one of the most characteristic feature of apoptosis. The results indicate that the use of STAR triggers apoptosis in cancer cells compared to various normal cells, what makes these nanoparticles a promising drug in therapeutic strategy, which targets apoptosis. We demonstrate highlighting potential of star polymers as an innovative tool for anti-cancer therapy.

Microorganization of ovaries and oogenesis of Haplotaxis sp. (Clitellata: Haplotaxidae).

Ovaries of Haplotaxis sp. were studied in active and nonactive states, that is, in a sexually mature specimen and in specimens outside of the reproductive period. Two pairs of ovaries were found in segments XI and XII. Especially in the nonactive state, they were in close contact with copulatory glands. Each ovary was composed of germ cells interconnected with syncytial cysts, which were enveloped by a layer of somatic cells. Within cysts each germ cell had one ring canal connecting it to the common anuclear cytoplasmic mass called a cytophore. During oogenesis clustering germ cells differentiated into nurse cells and oocytes; thus, the oogenesis was recognized as meroistic. Vitellogenic oocytes were detached from the ovaries and continued yolk absorption within the body cavity. Because recent studies have shown the variety of ovaries and germ line cyst organization in clitellates and suggest their evolutionary conservatism at the family or subfamily level, the data presented here can be valid in understanding the phylogenetic relationships among Clitellata. In this context, ovaries found in Haplotaxis sp. resembled those of the "Tubifex" type. "Tubifex" ovaries are characteristic for numerous microdrile taxa (tubificines, limnodriloidines, propappids, lumbriculids, and leech-like branchiobdellids) and can be regarded as the primary character for these Clitellata in which germ-line cysts are formed during early oogenesis. As the family Haplotaxidae is currently considered to be paraphyletic and the species studied here belongs to Haplotaxidae sensu stricto, our results support the close relationship of Haplotaxidae sensu stricto to the clade consisting of Lumbriculidae, Branchiobdellida, and Hirudinida, in which lumbriculids are sister to the latter two.

Fine structure of the midgut epithelium of Nicoletia phytophila Gervais, 1844 (Zygentoma: Nicoletiidae: Nicoletiinae) with special emphasis on its degeneration.

The midgut epithelium of Nicoletia phytophila is composed of columnar digestive cells and regenerative cells that form regenerative nests. The cytoplasm of midgut epithelial cells shows typical regionalization in organelle distribution. Two types of regenerative cells have been distinguished: cells which are able to divide intensively and cells which differentiate. Spot desmosomes have been observed between neighboring regenerative cells. The occurrence of intercellular junctions is discussed. The midgut epithelium degenerates both in an apoptotic and necrotic way. Necrosis proceeds during each molting period (cyclic manner), while apoptosis occurs between each molting, when the midgut epithelium is responsible for e.g. digestion. These processes of epithelium degeneration are described at the ultrastructural level. Our studies not only add new information about fine structure of the midgut epithelium of N. phytophila, but contribute to resolving the relationships within the Zygentoma. There are no doubts about the very close sister position of Nicoletiidae and Ateluridae. The midgut epithelium characters confirm their close relationship. However we do not recommend classifying the atelurid genera only within Nicoletiidae: Nicoletiinae.

Effects of short- and long-term exposure to cadmium on salivary glands and fat body of soil centipede Lithobius forficatus (Myriapoda, Chilopoda): Histology and ultrastructure.

Cadmium (Cd) is the most widely studied heavy metal in terms of food-chain accumulation and contamination because it can strongly affect all environments (e.g., soil, water, air). It can accumulate in different tissues and organs and can affect the organism at different levels of organization: from organs, tissues and cells though cell organelles and structures to activation of mechanisms of survival and cell death. In soil-dwelling organisms heavy metals gather in all tissues with accumulation properties: midgut, salivary glands, fat body. The aim of this study was to describe the effects of cadmium on the soil species Lithobius forficatus, mainly on two organs responsible for gathering different substances, the fat body and salivary glands, at the ultrastructural level. Changes caused by cadmium short- and long-term intoxication, connected with cell death (autophagy, apoptosis, necrosis), and the crosstalk between them, were analyzed. Adult specimens of L. forficatus were collected in a natural environment and divided into three experimental groups: C (the control group), Cd1 (cultured in soil with 80 mg/kg of CdCl2 for 12 days) and Cd2 (cultured in soil with 80 mg/kg of CdCl2 for 45 days). Transmission electron microscopy revealed ultrastructural alterations in both of the organs analyzed (reduction in the amount of reserve material, the appearance of vacuoles, etc.). Qualitative analysis using TUNEL assay revealed distinct crosstalk between autophagy and necrosis in the fat body adipocytes, while crosstalk between autophagy, apoptosis and necrosis in the salivary glands was detected in salivary glands of the centipedes examined here. We conclude that different organs in the body can react differently to the same stressor, as well as to the same concentration and time of exposure. Different mechanisms at the ultrastructural level activate different types of cell death and with different dynamics.

Characterisation of white and yellow eye colour mutant strains of house cricket, Acheta domesticus.

Two eye-colour mutant strains, white (W) and yellow (Y) of house cricket Acheta domesticus were established in our laboratory. We phenotyped and genotyped the mutants, performed genetic crossings and studied the eye structure and pigment composition using light and electron microscopy and biochemical analysis. We show that W and Y phenotypes are controlled by a single autosomal recessive allele, as both traits are metabolically independent. The analysis of the mutants`eye structure showed a reduced number of dark pigment granules while simultaneously, and an increased amount of light vacuoles in white eye mutants was observed. Significant differences in eye pigment composition between strains were also found. The Y mutant had a lower number of ommochromes, while the W mutant had a lower number of ommochromes and pteridines. This indicates that mutated genes are involved in two different, independent metabolic pathways regulating tryptophan metabolism enzymes, pigment transporter granules or pigment granule formation.

The ovary organization in the marine limnodriloidin Thalassodrilides cf. briani (Annelida: Clitellata: Naididae) resembles the ovary of freshwater tubificins.

In Thalassodrilides cf. briani, the paired ovaries are inconspicuous and polarized structures with developmental gradient of germ cells along their long axis. The about 300 germ cells in the ovary are consolidated into one syncytial cyst and each cell is connected to a common and branched mass of cytoplasm via one stable cytoplasmic bridge. The germ cells differentiate their fate into nurse cells and oocytes. Only one oocyte grows in a given time; it gathers cell organelles and yolk and then it detaches from the gonad. Nurse cells appear to support oocytes development by providing with at least cell organelles. Such observations suggest ovary meroism. T. cf. briani, belonging to limnodriloidin naidids, has the same ovary organization as the representatives of tubificin naidids studied to date. This supports the concept of sister-group relations between Limnodriloidinae and Tubificinae. A similar ovary morphology is also known in several other groups of "microdrile" oligochaetes, which shows that this ovary type is the most widespread among this taxa. Moreover, living in marine or brackish-water sediments, T. cf. briani do not show any significant differences in their ovary structure and oogenesis with freshwater tubificins. This indicates its conservative character independent of the animal life environment.

Micromorphology of ovaries and oogenesis in Grania postclitellochaeta (Clitellata: Enchytraeidae).

The genus Grania comprises over 70 species of exclusively marine clitellate annelids belonging to the family Enchytraeidae. Morphologically, this genus is well separated from other enchytraeids, with thick cuticles, anterior segments I-IV fused into a "head", chaetal bundles consisting only of one stout chaeta, and reduction of circular musculature. The aim of the present study is to describe the ovary organization and the course of oogenesis in Grania postclitellochaeta, and to compare it with other known systems of ovary organization and oogenesis in clitellate annelids, especially in enchytraeids. Generally, oogenesis in G. postclitellochaeta can be divided into two phases: (i) early stages of oogenesis, occurring within the paired ovaries - each ovary is similar to a bunch of grapes, where each 'lobe' is a germ-line cyst enveloped by flat somatic cells, and (ii) oogenesis proper, which takes place within the body lumen where each growing oocyte is accompanied by its own group of nurse cells. Germ cells are interconnected by cytoplasmic channels (intercellular bridges, ring canals) and form syncytial cysts. As in other clitellate annelids, the cyst center contains a common cytoplasm (cytophore) to which each cell is connected by one ring canal only. Initially, within the ovary, all interconnected cells develop synchronously and are morphologically similar. At the time when the cysts detach from the ovary, one of the interconnected cells begins to gather nutrients, grows and becomes an oocyte, whereas the rest of the cells (nurse cells) do not continue meiosis and instead seem to provide the oocyte with macromolecules and cell organelles. Analysis of serial sections reveals that cysts are always composed of 16 cells - one oocyte and fifteen nurse cells. A comparative analysis showed that almost all features of oogenesis in G. postclitellochaeta are similar to that in other representatives of Enchytraeidae (mainly Enchytraeus albidus), suggesting evolutionary conservation of the process across this family.

The structure of the desiccated Richtersius coronifer (Richters, 1903).

Tun formation is an essential morphological adaptation for entering the anhydrobiotic state in tardigrades, but its internal structure has rarely been investigated. We present the structure and ultrastructure of organs and cells in desiccated Richtersius coronifer by transmission and scanning electron microscopy, confocal microscopy, and histochemical methods. A 3D reconstruction of the body organization of the tun stage is also presented. The tun formation during anhydrobiosis of tardigrades is a process of anterior-posterior body contraction, which relocates some organs such as the pharyngeal bulb. The cuticle is composed of epicuticle, intracuticle and procuticle; flocculent coat; and trilaminate layer. Moulting does not seem to restrict the tun formation, as evidenced from tardigrade tuns that were in the process of moulting. The storage cells of desiccated specimens filled up the free inner space and surrounded internal organs, such as the ovary and digestive system, which were contracted. All cells (epidermal cells, storage cells, ovary cells, cells of the digestive system) underwent shrinkage, and their cytoplasm was electron dense. Lipids and polysaccharides dominated among reserve material of storage cells, while the amount of protein was small. The basic morphology of specific cell types and organelles did not differ between active and anhydrobiotic R. coronifer.