Ovary organization and ultrastructure in six species of Amynthas and Metaphire earthworms (Annelida, Crassiclitellata, Megascolecidae).

Ovaries in earthworms belonging to the family Megascolecidae are paired structures attached to the septum in the anterior part of the XIII segment. They are fan to rosette shaped with numerous rows of growing oocytes, known as egg strings, radiating from the ovary center towards the segmental cavity. The histological and ultrastructural ovary organization in megascolecids and the course of oogenesis remain unknown. The paper presents the results of light and electron microscopy analyses of ovaries in six megascolecid species, three from the genus Amynthas and three from Metaphire. Both parthenogenetic and sexually reproducing species were included in the study. The organization and ultrastructure of ovaries in all studied species are broadly similar. Considering the histological organization of ovaries, they could be divided into two zones. Zone I (proximal, close to the connection with the septum) is tightly packed with germline and somatic cells. Germ cells are interconnected via intercellular bridges and thin strands of the central cytoplasm (known as cytophore) and form syncytial cysts. Cysts unite oogonia, early meiotic cells (till diplotene), and clustering cells develop synchronously. During diplotene, interconnected cells lose developmental synchrony; most probably, one cell per cyst grows faster than others, detaches from the cysts, and becomes an oocyte. The remaining cells grow slightly and are still interconnected via the thin and reticular cytophore; these cells are considered nurse cells. Zone II has a form of egg strings where growing oocytes are isolated one from another by thin somatic cells and form short cords. We present the ultrastructural details of germline and somatic cells. We propose the term "Amynthas" type of ovaries for this ovary organization. We suppose that such ovaries are characteristic of other megascolecids and related families.

Ovary micromorphology in hormogastrid earthworms with a particular emphasis on the organization of the germline cysts.

There is a gap in our knowledge of microorganization and the functioning of ovaries in earthworms (Crassiclitellata) and allied taxa. Recent analyses of ovaries in microdriles and leech-like taxa revealed that they are composed of syncytial germline cysts accompanied by somatic cells. Although the pattern of cyst organization is conserved across Clitellata - each cell is connected via one intercellular bridge (ring canal) to the central and anuclear cytoplasmic mass termed the cytophore - this system shows high evolutionary plasticity. In Crassiclitellata, only the gross morphology of ovaries and their segmental localization is well known, whereas ultrastructural data are limited to lumbricids like Dendrobaena veneta. Here we present the first report about ovarian histology and ultrastructure in Hormogastridae, a small family of earthworms inhabiting the western parts of the Mediterranean sea basin. We analyzed three species from three different genera and showed that the pattern of ovary organization is the same within this taxon. Ovaries are cone-like, with a broad part connected to the septum and a narrow distal end forming an egg string. Ovaries are composed of numerous cysts uniting a small number of cells, eight in Carpetania matritensis. There is a gradient of cysts development along the long ovary axis, and three zones can be distinguished. In zone I, cysts develop in complete synchrony and unite oogonia and early meiotic cells (till diplotene). Then (zone II), the synchrony is lost, and one cell (prospective oocyte) grows faster than the rest (prospective nurse cells). In zone III, oocytes pass the growth phase and gather nutrients; at this time, their contact with the cytophore is lost. Nurse cells grow slightly, eventually die via apoptosis, and are removed by coelomocytes. The most characteristic feature of hormogastrid germ cysts is the inconspicuous cytophore in the form of thread-like thin cytoplasmic strands (reticular cytophore). We found that the ovary organization in studied hormogastrids is very similar to that described for D. veneta and propose the term "Dendrobaena" type of ovaries. We expect the same microorganization of ovaries will be found in other hormogastrids and lumbricids.

Uncovering nanotoxicity of a water-soluble and red-fluorescent [70]fullerene nanomaterial.

Engineered fullerene materials have attracted the attention of researchers in the biomedical sciences, especially when their synthetic methodology is developed to endow them with significant levels of water-solubility and bioavailability. In this study, we synthesized and characterized a water-soluble and red-fluorescent [70]fullerene nanomaterial, which fluoresced at 693 nm with a quantum yield of 0.065 and a large Stokes shift (around 300 nm). The fullerene nanomaterial generated mainly singlet oxygen after illumination with blue LED light, while superoxide anion radical production was minimal. The transmission electron microscopy as well as fluorescent studies of Drosophila melanogaster revealed that prepared [70]fullerene nanoparticles had better bioavailability than pristine [70]fullerene nanoparticles. The designed nanomaterials were observed in the apical, perinuclear, and basal regions of digestive cells, as well as the basal lamina of the digestive system's epithelium, with no damage to cell organelles and no activation of degenerative processes and cell death. Our findings provide a new perspective for understanding the in vivo behavior of fullerene nanomaterials and their future application in bioimaging and light-activated nanotherapeutics.

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.

Ovary micromorphology and oogenesis in a rhyacodriline oligochaete (Clitellata: Naididae, Rhyacodrilinae).

The main goal of the article is to describe the ovary organization and oogenesis in Peristodrilus montanus, an aquatic oligochaete of the subfamily Rhyacodrilinae. The presented analysis will not only enrich the knowledge about how eggs are formed but, because of the suggested conservatism of ovary organization in clitellate annelids, can contribute to disentangling the complex phylogenetic relationships of the rhyacodrilines within Naididae. The paired, conically shaped ovaries are located in segment XI. They are composed of a dozen or so syncytial germ-line cysts, which are associated with somatic cells. Each germ cell in a cyst has one intercellular bridge that joins it to a central and anuclear cytoplasmic mass, the cytophore. This pattern of cyst organization is typical for all clitellates that have been studied to date. Initially, the germ cells in a cyst undergo a synchronous development, however, there is no synchrony between cysts, and therefore there is a developmental gradient (oogonia, pre-diplotene germ cells, germ cells in diplotene) of oogenesis along the long ovary axis. The cysts are composed of a maximum of 32 cells. Cysts with cells in diplotene detach from the ovaries and the extraovarian phase of oogenesis begins. The developmental synchrony is lost, one cell (an oocyte) per cyst starts to gather cell components and yolk and grows considerably. The remaining cells grow to some extent and function as nurse cells. Like in other microdriles, P. montanus oocytes are rich in yolk; other features of oogenesis are also similar to those that are known from other microdrile taxa. The system of ovary organization found in the studied species is broadly similar to the corresponding features known from Naidinae and Phreodrilidae and, to some extent, in Enchytraeidae. However, this system is different from the one that is known in Tubificinae, Limnodriloidinae and Branchiurinae.

Morphology of Mitochondria in Syncytial Annelid Female Germ-Line Cyst Visualized by Serial Block-Face SEM.

Mitochondria change their morphology and distribution depending on the metabolism and functional state of a cell. Here, we analyzed the mitochondria and selected structures in female germ-line cysts in a representative of clitellate annelids - the white worm Enchytraeus albidus in which each germ cell has one cytoplasmic bridge that connects it to a common cytoplasmic mass. Using serial block-face scanning electron microscopy (SBEM), we prepared three-dimensional ultrastructural reconstructions of the entire selected compartments of a cyst at the advanced stage of oogenesis, i.e. the nurse cell, cytophore, and cytoplasmic bridges of all 16 cells (15 nurse cells and oocyte). We revealed extensive mitochondrial networks in the nurse cells, cytophore and mitochondria that pass through the cytoplasmic bridges, which indicates that a mitochondrial network can extend throughout the entire cyst. The dynamic hyperfusion state was suggested for such mitochondrial aggregations. We measured the mitochondria distribution and revealed their polarized distribution in the nurse cells and more abundant accumulation within the cytophore compared to the nurse cell. A close association of mitochondrial networks with dispersed nuage material, which seems to be the structural equivalent of a Balbiani body, not described in clitellate annelids so far, was also revealed.

Symplasmic isolation marks cell fate changes during somatic embryogenesis.

Cell-to-cell signalling is a major mechanism controlling plant morphogenesis. Transport of signalling molecules through plasmodesmata is one way in which plants promote or restrict intercellular signalling over short distances. Plasmodesmata are membrane-lined pores between cells that regulate the intercellular flow of signalling molecules through changes in their size, creating symplasmic fields of connected cells. Here we examine the role of plasmodesmata and symplasmic communication in the establishment of plant cell totipotency, using somatic embryo induction from Arabidopsis explants as a model system. Cell-to-cell communication was evaluated using fluorescent tracers, supplemented with histological and ultrastructural analysis, and correlated with expression of a WOX2 embryo reporter. We showed that embryogenic cells are isolated symplasmically from non-embryogenic cells regardless of the explant type (immature zygotic embryos or seedlings) and inducer system (2,4-dichlorophenoxyacetic acid or the BABY BOOM (BBM) transcription factor), but that the symplasmic domains in different explants differ with respect to the maximum size of molecule capable of moving through the plasmodesmata. Callose deposition in plasmodesmata preceded WOX2 expression in future sites of somatic embryo development, but later was greatly reduced in WOX2-expressing domains. Callose deposition was also associated with a decrease DR5 auxin response in embryogenic tissue. Treatment of explants with the callose biosynthesis inhibitor 2-deoxy-D-glucose supressed somatic embryo formation in all three systems studied, and also blocked the observed decrease in DR5 expression. Together these data suggest that callose deposition at plasmodesmata is required for symplasmic isolation and establishment of cell totipotency in Arabidopsis.

Microscopic analysis of spermatogenesis and mature spermatozoa in the amphibian leech Batracobdella algira (Annelida, Clitellata, Hirudinida).

Spermatogenesis and spermatozoa ultrastructure of the amphibian leech Batracobdella algira Moquin-Tandon, 1846 (Hirudinida: Glossiphoniidae) have been investigated by means of electron and fluorescent microscopy. In B. algira, there are seven pairs of testisacs (testes) that are located latero-ventrally throughout the body. Each testis contains numerous cysts with developing germ cells. The germ cells in a given cyst are in the same developmental stage (i.e., there are spermatogonial, spermatocytic, and spermatid cysts); however, there is no developmental synchrony between the cysts, and therefore, all of the developmental stages occur simultaneously in the same testis. In the cysts, each germ cell is connected to acentral cytoplasmic mass, the cytophore, by one intercellular bridge. The spermatozoa of the studied species conform to the general organization plan that is known for Hirudinida: they are filiform cells that are formed in sequence by an elongated and twisted acrosome that consists of an anterior and posterior acrosome, a fully condensed and helicoid nucleus, a midpiece composed of a single and twisted mitochondrion that is characteristically surrounded by an electron-dense sheath, and a flagellum with the conventional 9 × 2 + 2 axonemal pattern. Using a comprehensive approach, we compared our findings with the ultrastructural data that had been obtained from the spermatozoa of the other hirudinids that have been studied to date. Only minor differences in the length and shape of the studied organelles were found which seems to be connected with the different ways of insemination, specific properties of female reproductive tracts, and physiology of fertilization. Additionally, we studied the organization of the microtubular cytoskeleton in male germline cysts at consecutive stages of spermatogenesis using fluorescent and electron microscopy. By comparing the present data with those from Oligochaeta, Branchiobdellida, and Acanthobdellida, we found that only the presence of an anterior acrosome characterizes the true leeches and that, at present, should be regarded as an autapomorphic character of Hirudinida. Our results showed that the arrangement of the microtubules changed dynamically during spermatogenesis.

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.

Low mitochondrial activity within developing earthworm male germ-line cysts revealed by JC-1.

The male germ-line cysts that occur in annelids appear to be a very convenient model for spermatogenesis studies. Germ-line cysts in the studied earthworm are composed of two compartments: (1) germ cells, where each cell is connected via one intercellular bridge to (2) an anuclear central cytoplasmic mass, the cytophore. In the present paper, confocal and transmission electron microscopy were used to follow the changes in the mitochondrial activity and ultrastructure within the cysts during spermatogenesis. JC-1 was used to visualize the populations of mitochondria with a high and low membrane potential. We used the spot detection Imaris software module to obtain the quantitative data. We counted and compared the 'mitochondrial spots' - the smallest detectable signals from mitochondria. It was found that in all of the stages of cyst development, the majority of mitochondria spots showed a green fluorescence, thus indicating a low mitochondrial membrane potential (MMP). Moreover, the number of active mitochondria spots that were visualized by red JC-1 fluorescence (high MMP) drastically decreased as spermatogenesis progressed. As much as 26% of the total number of mitochondrial spots in the spermatogonial cysts showed a high MMP - 19% in the spermatocytes, 24% in the isodiametric spermatids and 3% and 6%, respectively, in the cysts that were holding early and late elongate spermatids. The mitochondria were usually thread-like and had an electron-dense matrix and lamellar cristae. Then, during spermiogenesis, the mitochondria within both the spermatids and the cytophore had a tendency to form aggregates in which the mitochondria were cemented by an electron-dense material.

Serial block face SEM visualization of unusual plant nuclear tubular extensions in a carnivorous plant (Utricularia, Lentibulariaceae).

Background and Aims: In Utricularia nelumbifolia , the nuclei of placental nutritive tissue possess unusually shaped projections not known to occur in any other flowering plant. The main aim of the study was to document the morphology and ultrastructure of these unusual nuclei. In addition, the literature was searched to find examples of nuclear tubular projections in other plant groups, and the nuclei of closely related species of Utricularia (i.e. sects Iperua , Orchidioides , Foliosa and Utricularia ) were examined. Methods: To visualize the complexity of the nuclear structures, transmission electron microscopy (TEM) was used, and 3-D ultrastructural reconstructions were made using the serial block face scanning electron microscopy (SBEM) technique. The nuclei of 11 Utricularia species, i.e. U. nelumbifolia , U. reniformis , U. cornigera , U. nephrophylla (sect. Iperua ), U. asplundii , U. alpina , U. quelchii (sect. Orchidioides ), U. longifolia (sect. Foliosa ), U. intermedia , U. minor and U. gibba (sect. Utricularia ) were examined. Key Results: Of the 11 Utricularia species examined, the spindle-like tubular projections (approx. 5 µm long) emanating from resident nuclei located in placental nutritive tissues were observed only in U. nelumbifolia . These tubular nuclear extensions contained chromatin distributed along hexagonally shaped tubules. The apices of the projections extended into the cell plasma membrane, and in many cases also made contact at the two opposing cellular poles, and with plasmodesmata via a short cisterna of the cortical endoplasmic reticulum. Images from the SBEM provide some evidence that the nuclear projections are making contact with those of neighbouring cells. Conclusions: The term chromatubules (chromatin-filled tubules) for the nuclear projections of U. nelumbifolia placental tissue was proposed here. Due to the apparent association with the plasma membrane and plasmodesmata, it was also speculated that chromatubules are involved in nucleus-cell-cell communication. However, further experimental evidence is required before any functional hypothesis can be entertained.

Analysis of the cytoskeleton organization and its possible functions in male earthworm germ-line cysts equipped with a cytophore.

We studied the organization of F-actin and the microtubular cytoskeleton in male germ-line cysts in the seminal vesicles of the earthworm Dendrobaena veneta using light, fluorescent and electron microscopy along with both chemically fixed tissue and life cell imaging. Additionally, in order to follow the functioning of the cytoskeleton, we incubated the cysts in colchicine, nocodazole, cytochalasin D and latrunculin A. The male germ-line cells of D. veneta are interconnected via stable intercellular bridges (IB), and form syncytial cysts. Each germ cell has only one IB that connects it to the anuclear central cytoplasmic mass, the cytophore. During the studies, we analyzed the cytoskeleton in spermatogonial, spermatocytic and spermatid cysts. F-actin was detected in the cortical cytoplasm and forms distinct rings in the IBs. The arrangement of the microtubules changed dynamically during spermatogenesis. The microtubules are distributed evenly in whole spermatogonial and spermatocytic cysts; however, they primarily accumulate within the IBs in spermatogonia. In early spermatids, microtubules pass through the IBs and are present in whole cysts. During spermatid elongation, the microtubules form a manchette while they are absent in the cytophore and in the IBs. Use of cytoskeletal drugs did not alter the general morphology of the cysts. Detectable effects-the occurrence of nuclei in the late spermatids and manchette fragments in the cytophore-were observed only after incubation in nocodazole. Our results suggest that the microtubules are responsible for cytoplasmic/organelle transfer between the germ cells and the cytophore during spermatogenesis and for the positioning of the spermatid nuclei.

An ultrastructural study of the ovary cord organization and oogenesis in Erpobdella johanssoni (Annelida, Clitellata: Hirudinida).

The aims of the present study were to analyze the ovary cord structure and oogenesis in Erpobdella johanssoni under light, fluorescent and transmission electron microscopy and to compare the obtained results with other clitellate annelids, especially with other arhynchobdellid leeches. Each of the paired ovaries is composed of the ovary wall (ovisac) and several (7-8) short, cone-shaped ovary cords. The ovary cords are of the "Erpobdella" type, i.e. they are short and polarized and five zones containing germ cells at consecutives stages of their development can be distinguished along their long axis. One, huge somatic cell (the apical cell), oogonia and premeiotic germ cells occur at the tip of the apical part of the ovary cord - zone I. Below, in zone II germ cells enter meiosis, whereas in zone III only a few cells continue meiosis and gather nutrients (oocytes), while the rest become nurse cells. In zone IV, huge vitellogenic oocytes form protuberances on the surface of the cord, and degenerating germ cells were observed at the base of the ovary cord (zones IV and V). The germline cells form syncytial cysts in zones I-III. The germline cysts have broadly the same architecture as in the ovaries of all of the clitellate annelids that have been described to date. Each germ cell in a cyst has only one cytoplasmic bridge connecting it to the common cytoplasmic mass - the cytophore. The cytophore is poorly developed, and it has the form of thin, long cytoplasmic strands. The presence of two categories of germ cells suggests a meroistic mode of oogenesis. The germline cysts are closely associated with somatic, follicular cells. There are two subpopulations of follicular cells: one envelops the growing oocytes, while the second is distributed between other germ cells. The entire ovary cord is additionally enveloped by a layer of somatic cells with a spongy appearance - the spongiosa cells. A characteristic feature of vitellogenic oocytes is the condensation of the chromosomes into a karyosome. Fully grown oocytes are excluded from the ovary cords and float freely in the ovisac lumen.