Cajal bodies (coiled bodies) in the nuclei of the house cricket (Acheta domesticus) oocytes.

The morphology and fine structure of Cajal bodies (coiled bodies, CB) in the germinal vesicles (oocyte nuclei) of the house cricket, Acheta domesticus have been analyzed. It is shown that in the studied species CBs arise as early as in the youngest previtellogenic oocytes, and are located next to or within aggregations of multiple nucleoli. Surprisingly, two morphological types of CBs have been found in the analyzed specimens. On the basis of EM studies we suggest that they represent subsequent developmental stages of CB morphogenesis.

The ovaries of Cicadomorpha: distribution of microfilaments and microtubules in terminal filament cells.

The ovaries of the investigated homopterans are telotrophicmeroistic and consist of several (7-21 ) ovarioles. Each ovariole is composed of three elements: an anteriorly localized terminal filament, a tropharium, and a posterior vitellarium. The latter comprises several developing ovarian follicles in a linear arrangement. The terminal filaments are relatively solid and composed of two distinct types of cells: the apical cells (ApCs) and the basal cells (BaCs). The BaCs are disc-shaped and oriented perpendicularly to the long axis of the ovariole, whereas the ApCs are strongly elongated and arranged parallel to this axis. The distribution of cytoskeletal elements has been studied with the use of electron microscope and histochemical methods. We show that the ApCs house prominent bundles of highly ordered microfilaments and/or parallel arranged microtubules. In contrast, BaCs contain only individual microtubules that are predominantly located in peripheral regions of the cells. It is suggested that microfilaments and microtubules present in the ApCs are responsible for the mechanical rigidity of the terminal filaments.

rDNA amplification in previtellogenic and vitellogenic oocytes of symphylans (Arthropoda, Myriapoda).

Tube-shaped ovaries of symphylans house numerous developing oocytes that are accompanied by somatic follicular cells. Oocyte nuclei (germinal vesicles) are relatively large and ovoid. During early previtellogenesis they contain compact spherical bodies and lampbrush chromosomes immersed in a translucent karyoplasm. Fluorescent labeling with DAPI and propidium iodide has revealed the presence of both DNA and RNA in the spherical bodies. As previtellogenesis advances, small RNA- and AgNOR-positive nucleoli bud off from these bodies. Full-grown nucleoli consist of coarse-granular material and comprise electron-transparent vacuoles. Our results suggest that in symphylan germinal vesicles amplification of rDNA genes takes place, and that the spherical bodies represent accumulations of extrachromosomal rDNA (rDNA bodies) after commencement of transcriptional activity.

Structure of ovaries and oogenesis in Corydalidae and Chauliodidae (Insecta, Megaloptera). I. Architecture of adult ovarioles and previtellogenesis.

The results of histological and EM studies on the ovaries of three representatives of Megaloptera: Chauliodes pectinicornis, Nigronia fasciata (Chauliodidae), and Corydalus peruvianus Corydalidae) are presented. It is shown that the ovaries of all 3 investigated species are panoistic (secondary panoistic, = neopanoistic) and consist of numerous (more than a hundred) ovarioles that are differentiated into 3 well-defined regions: the terminal filament, the germarium, and the vitellarium. The germaria of adult females are apparently non-functional and contain germ and somatic cells in various stages of degeneration. The vitellaria are composed of 12-15 developing ovarian follicles (= oocytes surrounded by follicular cells) in a linear arrangement. In adult females these follicles can be classified into early previtellogenic, late previtellogenic, vitellogenic, and choriogenic. During early previtellogenesis oocyte nuclei (= germinal vesicles) contain single nucleolar masses. Histochemical analyses indicate that within the masses DNA as well as AgNOR proteins are present. During subsequent stages of the previtellogenic growth nucleolar masses gradually break down into smaller aggregations of coarse granular material, i.e. multiple nucleoli. In chauliodids the nucleoli are distributed evenly throughout the nucleoplasm while in the corydalid, C. peruvianus, they form a characteristic ring. The presented results are discussed in a phylogenetic context.

Balbiani bodies in cricket oocytes: development, ultrastructure, and presence of localized RNAs.

Formation of two spherical Balbiani bodies along the long axis of previtellogenic oocytes in Acheta domesticus was demonstrated by differential interference microscopy. The structures form adjacent to and on opposite sides of the germinal vesicle, the anterior body first. Each migrates to the nearest pole of the elongating oocyte and retains its spherical structure until occluded from view by accumulating yolk. In situ hybridization, immunocytochemistry, and confocal immunofluorescent microscopy showed Balbiani body components to include y-tubulin, alpha-tubulin, EF1alpha, and several RNAs homologous to localized Xenopus RNAs implicated in embryonic axis formation or germ cell determination. The latter include Xcat2, Xwnt11, Xlsirt, and Xpat. Balbiani body ultrastructure includes a dense cloud of tubular mitochondria, rough ER, Golgi-like membrane aggregates, and microtubules. The results suggest that molecules and mechanisms specifying early determinative events for embryogenesis in vertebrates and insects are highly conserved and that Balbiani bodies may have a role in establishing developmental asymmetry in the cricket.

Structure of ovarioles in adult queens and workers of the common wasp, Vespula germanica (Hymenoptera: Vespidae).

The ovaries of the common wasp, Vespula germanica are polytrophic-meroistic and consist of 2-3 (workers) or 7 (queens) ovarioles. The ovarioles are differentiated into three regions: a terminal filament, a germarium, and a vitellarium. The germaria of both castes consist of two zones: an anterior zone of germ-cell cluster formation and a posterior one of germ-cell cluster differentiation. The vitellaria comprise 4-6 (workers) or 7-10 (queens) ovarian follicles (egg chambers). Each chamber consists of an oocyte and about 60 isodiametric nurse cells (trophocytes). The egg chambers have been arbitrarily classified into four developmental categories: early and late previtellogenic, vitellogenic, and choriogenic. The process of oogenesis in workers proceeds only up to the onset of the late previtellogenesis. Neither vitellogenic nor choriogenic egg chambers were observed in this caste. During early and late previtellogenesis the envelope of the oocyte nucleus proliferates and becomes highly folded. This process leads to the formation of characteristic organelles, termed accessory nuclei (AN). Although AN arise in the oocytes of both queens and workers, their number in the latter caste is always considerably lower. At the onset of the late previtellogenesis AN start to migrate towards the periphery of the oocyte where they reside till the end of oogenesis. The physiological state of the worker ovaries is discussed in the light of the presented results.

Ultrastructure and function of nurse cells in phthirapterans. Possible function of ramified nurse cell nuclei in the cytoplasm transfer.

The structure of nurse cells as well as the distribution of cytoskeletal elements (actin filaments, microtubules) in three representatives of phthirapterans: the pig louse, Haematopinus suis (Anoplura) and bird lice, Eomenacanthus stramineus, Columbicola columbae (Mallophaga) were investigated. All three species have polytrophic-meroistic ovaries which means that each oocyte remains connected with a group of nurse cells via specialized cytoplasmic canals-intercellular bridges (ring canals). Throughout vitellogenesis, various macromolecules as well as organelles (mitochondria, endoplasmic reticulum vesicles, ribosomes) are transferred from the nurse cells to the oocyte. During this flow, the nurse cell nuclei do not enter the oocyte and are retained in the cell centers. In holometabolous insects (e.g. Drosophila, hymenopterans), the central position of nurse cell nuclei is maintained by cytoskeletal elements (actin filaments or microtubules). In the investigated species, the nurse cells are equipped with large, highly extended (irregularly lobed) nuclei. The inner nuclear membrane is lined with a relatively thick layer of nuclear lamina. Ultrastructural analysis and staining with rhodamine-labeled phalloidin revealed that the nurse cell cytoskeleton is poorly developed and represented only by: (1) single microtubules in the perinuclear cytoplasm; and (2) the F-actin layer in the cortical cytoplasm. In the light of this, we postulate that in phthirapterans the position of nurse cell nuclei during the cytoplasm transfer is maintained not by the cytoskeletal elements, but by a largely extended shape of the nuclei (i.e. their elongated extensions).

Organization and possible functions of microtubule cytoskeleton in hymenopteran nurse cells.

The results of systematic cytochemical and EM studies on the distribution of actin filaments and microtubules in hymenopteran nurse cells are presented. We demonstrate that each nurse cell nucleus is surrounded by a thick three-dimensional cage of microtubules that is engaged in maintaining the position of the nuclei in the cell centers during the flow of the cytoplasm from nurse cells into the oocyte. Hence, the cages represent functional counterparts of actin bundles described in the Drosophila nurse cells. Furthermore, our data suggest that a subset of the microtubules is involved in transferring nuage aggregates from the vicinity of the nucleus towards the nurse cell periphery and the nearest intercellular bridge. A conclusion is reached that despite similar polytrophic organization of the ovaries in both hymenopterans and dipterans, the physiology of their nurse cell-oocyte syncytia appears distinctly different.

Neuropteroidea--different ovary structure in related groups.

Three different ovariole types have been described in the Neuropteroidea. In this review, comparative analysis of their structure and function is presented, and the results are used for phylogenetic considerations. Neuropteran polytrophic ovaries exhibit deviations from the basic polytrophic pattern found in other insects. Asynchronous divisions of germ cells result in a variable and unfixed number of cystocytes per cluster. In contrast to the typically branched system, spatial organization of the cystocyte connections in neuropteran egg chambers is almost linear. A more precise comparative study of ovariole structure and function within Neuroptera brings further support for the placement of Coniopterygidae as an early off-shoot from the main neuropteran phylogenetic lineage. Ovaries of Raphidioptera and Megaloptera: Sialidae represent a distinct type of telotrophic organization. Its almost identical character in both groups favours the concept on the origin of this telotrophy from the common ancestral polytrophic condition. Ovarioles of Megaloptera: Corydalidae are neopanoistic and it is emphasized here that this organization must have evolved independently from the polytrophic background. A hypothesis on paraphyletic origin of Megaloptera is thus supported.

Diptera--ovary structure and oogenesis in midges and flies.

Comparative study of ovary development and oogenesis in the dipterans revealed significant differences between the Nematocera (lower dipterans, midges) and the Brachycera (true flies). The occurrence of these differences emphasizes well the phylogenetic division of the Diptera into these major subgroups. Basic discrepancies were found in the course of ovary development and in the mode of follicular cell differentiation. In contrast to more advanced flies, in midges the initial stages of germ cell differentiation, i.e. divisions of gonial cells, germ cell cluster formation and diversification of cystocytes within clusters take place exclusively in the larval and early pupal stages. Moreover, the formation of cystocyte clusters precedes that of ovarioles. Differences in the behaviour of some follicular cells found between the ovarian follicles of midges and advanced flies suggest that both major dipteran subgroups may differ in the scenario and/or the mechanisms of terminal signalling leading to the determination of the anteriormost part of the body.

The ovaries of Mecoptera: basic similarities and one exception to the rule.

Two entirely different types of ovaries (ovarioles) have been described in mecopterans. In the representatives of Meropeidae, Bittacidae, Panorpodidae and Panorpidae the ovarioles are of the polytrophic-meroistic type. Four regions: a terminal filament, germarium, vitellarium and ovariole stalk can be distinguished in the ovarioles. The germaria house numerous germ cell clusters. Each cluster arises as a result of 2 consecutive mitoses of a cystoblast and consists of 4 sibling cells. The oocyte always differentiates from one of the central cells of the cluster, whereas the remaining 3 cells develop into large, polyploid nurse cells. The vitellaria contain 7-12 growing egg chambers (= oocyte-nurse cell complexes). In contrast, the ovaries of the snow flea, Boreus hyemalis, are devoid of nurse cells and therefore panoistic (secondary panoistic). The ovarioles are composed of terminal filaments, vitellaria and ovariole stalks only; in adult females functional germaria are absent. Histochemical tests suggest that amplification of rDNA takes place in the oocyte nuclei. Resulting dense nucleolar masses undergo fragmentation into multiple polymorphic nucleoli. The classification of extant mecopterans as well as the phylogenetic relationships between Mecoptera and Siphonaptera are discussed in the context of presented data.

Microtubules are responsible for the asymmetrical distribution of organelles in the oocytes of the hymenopteran, Chrysis ignita.

Previous morphological investigations have shown that the distribution of cellular organelles and reserve materials in hymenopteran oocytes is uneven and often gradient-like. It has been suggested that the microtubular cytoskeleton is responsible for creating and/or maintaining the distribution gradients. To test this hypothesis, adult females of the hymenopteran, Chrysis ignita were treated with a microtubule-assemble inhibitor, colchicine. The experiment resulted in a number of abnormalities observed in the egg chambers: the oocytes were devoid of organelle-free periplasm and oosome; cellular organelles and reserve materials were distributed randomly within the oocyte; nurse cells organization was significantly changed. These observations have confirmed a key role of microtubules in forming and/or maintaining the asymmetry of ooplasm in Chrysis ignita.

Egg shells of mallophagans and anoplurans (Insecta: Phthiraptera): morphogenesis of specialized regions and the relation to F-actin cytoskeleton of follicular cells.

The egg shells of investigated phthirapterans consist of three basic elements: an anterior operculum, a main egg shell and a posterior hydropyle. In some species these elements show further regional specializations: bristles and projections that facilitate attachment to feathers of the host, micropyles and aeropylar openings. All of the egg shell specializations are formed by distinct subpopulations of follicular cells. Staining with rhodamine-conjugated phalloidin has revealed that these subpopulations significantly differ in the distribution of microfilaments (F-actin). In this respect four morphological categories of the follicular cells have been distinguished: (1) cells devoid of processes and microvilli, with basal arrays of microfilaments, responsible for the secretion of a flat chorion; (2) cells devoid of processes and microvilli, separated by intercellular spaces, with basal arrays of microfilaments, responsible for the secretion of attachment structures; (3) cells equipped with actin-containing processes, responsible for the formation of micropyles or aeropyles, and (4) cells equipped with bundles of microvilli, responsible for the formation of hydropyles.

A new version of the Ag-NOR technique. A combination with DAPI staining.

The Ag-NOR staining technique is widely used for visualizing nucleolar organizer regions (NORs) in various plant and animal tissues. We describe a simple and time-saving combination of Ag-NOR staining with DNA detection by fluorescence microscopy. This modification was tested on cultured cells and semi-thin sections of plastic-embedded tissues. Of the different fixatives and embedding media used in our studies, the best results (i.e., high selectivity of staining, and lack of or very low background precipitation) were obtained with fixation in methanol-acetone at-20 degrees C for cultured cells, and fixation in 4% formaldehyde followed by embedding in Histocryl resin for tissue sections. The optimal time of Ag-NOR staining was determined experimentally for all materials tested. The specificity of the staining was checked at the electron microscopical level. Especially good results were obtained by mixing epifluorescence with standard bright-field illumination. In such a combination, Ag-NOR-positive nucleoli, or their fibrillar centres and dense fibrillar components, were clearly visible against a bright background of nuclear DNA.

Extrachromosomal amplification of rDNA in oocytes of Hemerobius spp. (Insecta, Neuroptera).

In previtellogenic oocytes of the neuropteran, Hemerobius spp., two distinct, DNA-positive intranuclear structures have been observed. Chromosomes of meiotic prophase assemble in the center of the oocyte nucleus forming a highly polymorphic karyosphere, which persists in this position until the very late stages of vitellogenesis. The extrachromosomal DNA body, containing amplified ribosomal genes, undergoes fragmentation and dispersion in the nucleoplasm. At the onset of previtellogenic growth, transcription of extra rDNA starts, which is accompanied by the appearance of dense, granular material (multiple nucleoli). Arising nucleoli gradually fill the nucleoplasm. At the electron microscopic (EM) level two electron dense structural forms of the granular material have been described. Together with general histological and ultrastructural analysis the amplification of rDNA genes in Hemerobius spp. oocytes has been demonstrated by means of the spreading technique, which has shown that extra rDNA is organized in rings containing various numbers of active ribosomal genes. The transcription activity of amplified genes is manifested in the form of typical "Christmas tree" structures.

Visualization of accessory nuclei from oocytes of the sawfly Athalia rosae (Hymenoptera: Tenthredinidae) by a spreading technique.

Oocytes of hymenopterans are equipped with specific organelles termed accessory nuclei (AN). These organelles are surrounded by a double envelope and contain RNA-positive inclusions. We have employed the spreading technique to analyse AN isolated from the ooplasm of the sawfly Athalia rosae. In electron microscopic spreading preparations AN appear as membranous, extensively folded structures. In their vicinity, numerous nuclear pore complexes have been found. This observation confirms the presence of pores in the envelopes of accessory nuclei.

Accessory nuclei in the oocytes of the cockoo wasp, Chrysis ignita (Hymenoptera: Aculeata).

In the oocytes of Chrysis ignita, accessory nuclei (AN) arise before yolk formation, during advanced previtellogenesis. Two categories of AN, small (SAN) and large (LAN) can be distinguished at this stage of development. Both AN categories contain two dense, morphologically distinct inclusions (termed A and B) immersed in a translucent matrix. AN are exclusively located in the cortical (peripheral) ooplasm. Moreover, LAN are asymmetrically distributed also along the antero-posterior axis of the oocyte, and occur preferentially in the anterior pole periplasm. In contrast, the distribution of SAN is uniform throughout the periplasm.

Ultrastructural studies on accessory nuclei in developing oocytes of the crustacean, Siphonophanes grubei.

Numerous accessory nuclei (AN) occur within peripheral cytoplasm of Siphonophanes grubei oocytes. They exhibit typical nuclear structure, with double membrane envelope pierced by pore complexes. Although no explicit evidence was shown, indirect arguments exist indicating that AN originate from the oocyte nucleus. Presence of clumps of DNP material within AN suggests that AN might play some role in amplified DNA metabolism. Participation of AN in cellular membrane elaboration is also postulated.

Unusual organization of the tropharium in the telotrophic ovarioles of an insect, Saldula saltatoria.

The tropharium of the common shorebug Saldula saltatoria consists of 2 zones: the apical mitotic region and the distal one comprising numerous mononucleate nurse cells. Each individual nurse cell is connected to the centrally located trophic core by a thin cytoplasmic projection referred to as a trophic process. The accumulations of a dense material interpreted as the remnants of intercellular bridge rim are observed associated with the trophic process membrane. In the light of these results the establishment of telotrophic ovarioles in hemipterans is discussed.