The ovary of Tubifex tubifex (Clitellata, Naididae, Tubificinae) Is composed of one, huge germ-line cyst that is enriched with cytoskeletal components

Recent studies on the ovary organization and oogenesis in Tubificinae have revealed that their ovaries are small polarized structures that are composed of germ cells in subsequent stages of oogenesis that are associated with somatic cells. In syncytial cysts, as a rule, each germ cell is connected to the central cytoplasmic mass, the cytophore, via only one stable intercellular bridge (ring canal). In this paper we present detailed data about the composition of germ-line cysts in Tubifex tubifex with special emphasis on the occurrence and distribution of the cytoskeletal elements. Using fixed material and live cell imaging techniques, we found that the entire ovary of T. tubifex is composed of only one, huge multicellular germ-line cyst, which may contain up to 2,600 cells. Its architecture is broadly similar to the cysts that are found in other clitellate annelids, i.e. a common, anuclear cytoplasmic mass in the center of the cyst and germ cells that are connected to it via intercellular bridges. The cytophore in the T. tubifex cyst extends along the long axis of the ovary in the form of elongated and branched cytoplasmic strands. Rhodamine-coupled phalloidin staining revealed that the prominent strands of actin filaments occur inside the cytophore. Similar to the cytophore, F-actin strands are branched and they are especially well developed in the middle and outermost parts of the ovary. Microfilaments are also present in the ring canals that connect the germ cells with the cytophore in the narrow end of the ovary. Using Tubulin-Tracker, we found that the microtubules form a prominent network of loosely and evenly distributed tubules inside the cytophore as well as in every germ cell. The well-developed cytoskeletal elements in T. tubifex ovary seem to ensure the integrity of such a huge germline cyst of complex (germ cells - ring canals - cytophore) organization. A comparison between the cysts that are described here and other well-known female germ-line cysts is also made

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 havebeen 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. Themidgut epitheliumdegenerates 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

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

Verification of Hypsibius exemplaris Gąsiorek 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. Introductio

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

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 M, 500 M, and 750 M 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

Influence of soil contaminated with cadmium on cell death in the digestive epithelium of soil centipede Lithobius forficatus (Myriapoda, Chilopoda)

Cadmium is a heavy metal that is treated as an environmental pollutant (air, water, soil). In order to understand the potential effects of cadmium in soil and soil invertebrates, it is important to describe all alterations which appear at different levels in organisms. The main aim of this study was to investigate, analyze and describe the alterations caused by cadmium short- and long-term intoxication at different levels in the organisms: from tissues to cells and organelles. In addition, the activation of cell deathmechanisms that take part in homeostasismaintenance according to cadmium has been studied. Therefore, as the species for this project, a terrestrial and well-known widespread European species – the centipede Lithobius forficatus (Myriapoda, Chilopoda, Lithobiomorpha) – was chosen. This omnivorous species lives under upper layers of soil, under stones, litter, rocks, and leaves, and it is also commonly found in human habitats. The animals were divided into three groups: C – the control group, animals cultured in a horticultural soil; Cd1 – animals cultured in a horticultural soil supplemented with 80 mg/kg (dry weight) of CdCl2, 12 days – short-term exposure; Cd2 – animals cultured in a horticultural soil supplemented with 80 mg/kg (dry weight) of CdCl2, 45 days – long-term exposure. The midgut was isolated from each specimen and it was prepared for analysis using some histological, histochemical and immunohistochemical methods. Our studies showed that short-term intoxication causes intensification of autophagy and digestion of reserve material, while long-term exposure to this heavy metal causes activation of cell death processes together with inhibition of autophagy connected with the lack of reserve material. Additionally, we can infer that autophagy and cell death are nutrient deprivation-induced processes. Finally, we can conclude that short- and long-term exposure of soil centipede to cadmium affects different mechanisms and processes of cell death

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

Effects of cadmium on mitochondrial structure and function in different organs: studies on the soil centipede Lithobius forficatus (Myriapoda, Chilopoda)

Mitochondria are organelles that play a crucial role in cell physiology, cell death, and aging. They are among the first responders to different stressors that originate from the environment. Cadmium as a heavy metal affects different levels of body organization: from organs through tissues and cells to organelles. Based on our previous research results, we decided to check how the exposure to cadmium affects the functioning of mitochondria in different organs of soil living centipede Lithobius forficatus. The activity of mitochondria in somatic and germ cells has been analyzed using transmission electron microscopy (TEM), confocal microscopy, and flow cytometry. Changes in the mitochondrial membrane potential and mitochondrial dismutase (MnSOD) activity in relation to the accumulation of reactive oxygen species (ROS) caused by cadmium exposure have been studied. Individuals were divided into 3 experimental groups depending on cadmium concentration in soil. Changes in mitochondrial ultrastructure caused by cadmium are tissuedependent and associated with an increase of ROS levels. The system of ROS and MnSOD activation works more efficiently in the case of gonads than in the digestive system. While the short-term cadmium exposure alters the fine structure of both the somatic and germ-line cells in gonads, the long-term cadmium exposure causes mitochondrial ultrastructure regeneration