Nuclear structures in the telotrophic ovarioles of nocturnal ground beetles (Tenebrionidae, Polyphaga). III. The oocyte nucleus. Electron microscopic data]

The fine structural organization of nuclei was studied in the growing oocytes of Blaps lethifera, B. mortisaga and Gnaptor spinimanus. In the beginning of diplotene the nuclei contain primary fibrillar nucleoli and numerous electron dense globules dispersed all over the nucleus; the loose chromosome material (lampbrush chromosomes) is distributed all over the nucleus. With the oocyte growth the chromosomes are spiralized and join into the karyosphere. A capsule of fibrous material forms around the karyosphere. The karyosphere nucleoli appear on the chromosomes and, then, move to the capsule region and outside its limits, to the nuclear envelope. They are fibrillar and non-active with respect to RNA synthesis. The fibrous material of the capsule is represented by strands which consist of bundles of cross-striated filaments. These latter contact directly with the chromosomes in the karyosphere and with the surface of the karyosphere nucleoli. The fibrillar-granular bodies are distributed along the strands in the capsule; they contain both RNA and DNA. The nature of extrachromosomal DNA in the karyosphere capsule and its participation in the formation of the capsule material are discussed. A suggestion is put forward on the similarity of the capsule strands with the modified central elements of synaptinemal complex.     

Nuclear bodies in the oocyte nucleus of ground beetles are enriched in snRNPs

Within the oocyte nucleus of many insect species, a variable number of intensely stained spherical bodies occur. These nuclear bodies differ significantly from nucleoli and their precise role in nuclei has not been elucidated yet. I have examined some of the histochemical properties as well as the molecular composition of these structures in a representative of ground (carabid) beetles. I demonstrate, using molecular markers, that the nuclear bodies are composed of small nuclear RNAs and associated proteins, including p80 coilin. Hence, they correspond to Cajal bodies (= coiled bodies) described in somatic cell nuclei as well as oocyte germinal vesicles in plant and animal organisms. It is suggested that Cajal bodies in the carabid germinal vesicle serve as a storage site for splicing factors.     

Structure and development of ovaries in the weevil,Anthonomus pomorum (Coleoptera,Polyphaga). II. Germ cells of the trophic chamber

The analysis of the germ cell cluster formation in Anthonomus pomorum (Coleoptera, Polyphaga, Curculionidae) has revealed that both linear and branched clones of cystocytes occur in the pupa stage. In the branched clones a poorly developed polyfusome is formed and cystocytes with maximally 3 intercellular bridges were found. In the linear clones the polyfusomes are absent. Further divisions of cystocytes produce exclusively linearly arranged cells. Just after metamorphosis (Imago-A stage), the process of the germ cell membrane reduction starts. Only 2 groups of cells retain cell membranes: i.e the most anteriorly localized group of cystocytes and the posteriorly located presumptive oocytes. The former cells divide mitotically during the summer. As a result an anterior-posterior gradient of the syncytialization process arises in the Imago-B stage (females preparing for hibernation). In the sexually mature females (Imago-C) the trophic chamber consists of a huge syncytial area with numerous nurse cell nuclei embedded in a common cytoplasm, and posteriorly located young oocytes surrounded by prefollicular cells. In the light of recent hypothesis concerning the germ cell cluster formation and telotrophy anagenesis in Polyphaga the significance of the presented results is discussed.     

Germ-cell cluster formation in the telotrophic meroistic ovary of <Emphasis Type="Italic">Tribolium castaneum</Emphasis> (Coleoptera,Polyphaga, Tenebrionidae) and its implication on insect phylogeny

Tribolium castaneum has telotrophic meroistic ovarioles of the Polyphaga type. During larval stages, germ cells multiply in a first mitotic cycle forming many small, irregularly branched germ-cell clusters which colonize between the anterior and posterior somatic tissues in each ovariole. Because germ-cell multiplication is accompanied by cluster splitting, we assume a very low number of germ cells per ovariole at the beginning of ovariole development. In the late larval and early pupal stages, we found programmed cell death of germ-cell clusters that are located in anterior and middle regions of the ovarioles. Only those clusters survive that rest on posterior somatic tissue. The germ cells that are in direct contact with posterior somatic cells transform into morphologically distinct pro-oocytes. Intercellular bridges interconnecting pro-oocytes are located posteriorly and are filled with fusomes that regularly fuse to form polyfusomes. Intercellular bridges connecting pro-oocytes to pro-nurse cells are always positioned anteriorly and contain small fusomal plugs. During pupal stages, a second wave of metasynchronous mitoses is initiated by the pro-oocytes, leading to linear subclusters with few bifurcations. We assume that the pro-oocytes together with posterior somatic cells build the center of determination and differentiation of germ cells throughout the larval, pupal, and adult stages. The early developmental pattern of germ-cell multiplication is highly similar to the events known from the telotrophic ovary of the Sialis type. We conclude that among the common ancestors of Neuropterida and Coleoptera, a telotrophic meroistic ovary of the Sialis type evolved, which still exists in Sialidae, Raphidioptera, and a myxophagan Coleoptera family, the Hydroscaphidae. Consequently, the telotrophic ovary of the Polyphaga type evolved from the Sialis type. Electronic supplementary material Supplementary material is available in the online version of this article at and is accessible for authorized users.     

Ultrastructure of the ovary of Dermatobia hominis (Diptera: Cuterebridae). II. Origin of the tunica propria in ovarioles

Ovaries up to the 8th day pupae of Dermatobia hominis were studied by transmission electron microscopy. Ovarioles were recognized in ovaries of 4-day old pre-pupae, surrounded by a thin tunica propria of acellular fibrillar material similar in structure to the internal portion of the external tunica of the ovary. There is continuity of the tunica propria and the ovarian tunica, indicating that the former structure originates from the tunica externa. In 5 to 7-day pupae the interstitial somatic cells from the apical region of the ovary, close to the ovarioles, show delicate filamentous material inside of their rough endoplasmic reticulum cisternae; similar material is seem among these cells. Our observations suggest that interstitial somatic cells do not originate the tunica propria but contribute to its final composition.     

Cytogenetics of the darkling beetles Zophobas aff. confusus and Nyctobates gigas (Coleoptera, Tenebrionidae)

Males of Zophobas aff. confusus and Nyctobates gigas (Tenebrionidae) collected in the State of Pernambuco, Brazil, were studied through conventional staining, C-banding, silver nitrate impregnation (AgNO(3)), and the base specific fluorochromes CMA(3) and DAPI. Z. aff. confusus was found to have 2n = 20 (9+Xyp) while N. gigas exhibited 2n = 18 (8+neoXY). Large pericentromeric blocks of constitutive heterochromatin (CH) were detected throughout the autosomal complement of the two species, except in one autosomal pair of N. gigas in which no heterochromatic block was observed. The sex chromosomes of both species were almost totally heterochromatic. Double staining with CMA(3)/DA (distamycin) and DAPI/DA marked CH in Z. aff. confusus. However, DAPI staining was more intense. N. gigas was found to possess blocks of CH-positive CMA(3) and homogeneous DAPI. AgNO(3) staining also revealed differences between the two species. In Z. confusus an NOR was observed in the sexual bivalent Xyp and N. gigas was found to have an autosomal NOR.     

Spatial structure of community of darkling beetles (Coleoptera: Tenebrionidae) in the Negev Highlands, Israel

The distribution of niches in resource space of darkling beetles of Negev desert, Israel were studied using discriminant analysis Seventeen environmental variables (substrate structure and vegetation) were measured at 375 plots within 15 grids The first three discriminant axes account for 77% of the variance and reflect a soil structure gradient from sand to clay (the first axis), a gradient of increasing productivity (the second axis), and a type of vegetation architecture (the third axis) Abundance and biomass of tenebrionids increased linearly along the first, the second and the third axes Species richness and diversity had a slanting parabola shape along the substrate axis and increased along the productivity axis There were two distinct spatial guilds of tenebrionid species One guild was composed by species that preferred sandy-gravel soils, and another guild consisted of species that preferred clay soils Niche overlapping of the "sandy-gravel" species was lower than that of the "clay" species Niches of the "sandy-gravel" species were randomly distributed in the resource space Those of the "clay" species formed a tight cluster with most niches placed in the center of the space, representing the centrifugal type of organization The conformity of the guild structure with the different types of organization was supported by compansons between empirical data and stochastic models The border between these two guilds matches with proposed earlier biogeographical boundary (Ramon-Zohar line)     

The analysis of data in studies of invertebrate reproduction. II. The analysis of oocyte size/frequency data,and comparison of different types of data

The measurement of oocyte sizes is the best method for examining reproductive cycles in the animals on which we have worked (mainly echinoderms and molluscs). The examination of standardised residuals in an R × C contingency table permits the detailed study of the growth of oocytes, and of differences in developmental stage both within and between samples. A nested design analysis of variance permits the quantification of the degree of seasonality in a species, and makes interspecific comparisons in the amount of inter- and intra-individual synchrony possible.

Comparisons between different types of data and methods of studying interpopulation differences in reproductive pattern are also reviewed.     

Structure and development of ovaries in the weevil, Anthonomus pomorum (Coleoptera, Polyphaga). I. Somatic tissues of the trophic chamber

In developing ovarioles of Anthonomus pomorum (Coleoptera, Polyphaga, Curculionidae) the trophic chambers (tropharia) are relatively large and consist of clusters (clones) of germ cells and various somatic tissues. Each ovariole is enclosed within an outer epithelial sheath (tunica externa). Throughout the pupal phase, the growth of this sheath is accelerated and precedes the development of the rest of the ovariole. As a result, the epithelial sheath proliferates anteriorly and forms an elongated "sleeve" that during the later stages of development becomes gradually filled by the growing tropharium. In the early pupal stage, a few terminal filament cells are observed in contact with the anterior end of the tropharium. These cells are separated from the rest of the trophic chamber by a transverse septum, which maintains continuity with the basal lamina. Beneath the basal lamina there is a layer of inner sheath cells, whereas inside the tropharium there are interstitial cells. These two types of cell differ morphologically in a mature ovary but they retain, until the end of the imago-B stage, a similar ultrastructure testifying to their common origin. At the posterior end of the tropharium, from the imago-B stage on, many young oocytes, surrounded by prefollicular cells, are observed. This is the so-called neck region of the tropharium. Extraction with Triton X-100 detergent showed that in a mature trophic chamber there are only individual microtubules arranged along the projections of interstitial cells. This indicates that the cytoskeleton elements (microfilaments and microtubules) participate only to a very limited extent in the spatial organisation of the tropharium in A. pomorum.     

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.     

Antispectrin antibodies stain the oocyte nucleus and the site of fertilization channels in the egg of Discoglossus pictus (Anura).

In Discoglossus pictus eggs, only the dimple contains ionic channels active at fertilization; in particular, chloride channels are found in the central portion of the dimple, which is also the site of sperm penetration. Moreover the dimple hosts an imposing cytoskeleton, consisting of a cortical network and bundles of microfilaments extending from the microvilli. Since spectrin cross links actin and is connected through ankyrin to anion transporters in the plasma membrane of erythrocytes as well as to anion channels in other cells, we studied, in D. pictus egg, the relationship between the localization of spectrin and the high polarization of ionic channels and cytoskeletal organization. By means of immunocytochemistry, we localized spectrin exclusively in the egg dimple. In an attempt to trace back the source of spectrin localization, we immunostained sections of D. pictus ovary and localized spectrin in the nuclei of previtellogenic oocytes, where actin is also present. Antispectrin staining remained until germinal vesicle breakdown. By contrast, a cortical localization was found only when the oocytes divided into two hemispheres and into the germinative area (GA), which, after germinal vesicle breakdown, gives rise to the dimple. At this stage the antispectrin signal was particularly strong in the GA. Using Rho-pialloidin, we also established that spectrin is generally present where F-actin is found. However, spectrin and F-actin do not have the same pattern of fluorescence. In conclusion, our data suggest that spectrin may play a role in oocyte and egg polarity. In eggs, it could be instrumental in anchoring to the cytoskeleton membrane proteins such as receptors and ionic channels, including chloride-permeable channels.     

Nuclear structures in the late oogenesis of Rana temporaria. III. Electron microscopic studies]

The ultrastructural organization of the vitellogenic oocyte nucleus (stage VI, according to Duryee, 1950) was studied in normal and in vitro hormone-stimulated maturing oocytes of Rana temporaria. At this stage, numerous nucleoli are gathered around the knot of highly contracted chromosomes (the karyosphere) thus making the karyosphere capsule. Light microscope observations reveal three zones in the capsule: a central fibrous zone separating the chromosomes from the nucleoli, a middle zone, consisting of numerous nucleoli and a distinct fibrous componen; in addition a fibrous zone on the capsule periphery is seen. The nucleoli are fibrillar, bear no proribosomal granules and do not synthesize RNA. This period is characterized by an intensive fragmentation and segregation of the nucleolar material. Numerous micronucleoli and nuclear bodies occur in the nucleus. The nucleoli are normally compound and irregular in shape to become spherical in hormone-stimulated maturing oocytes. In the central fibrous zone of the capsule, separating the chromosomes from the nucleoli, some peculiar abundant accumulations of annuli were detected lacking the membranes component. Annuli are linked with the fibrous material and are regularily packed making peculiar pseudomembranes (PMM). The chromatin is connected with PMM directly. In the middle zone of the capsule, accumulations of PMM are also seen, though less abundant and less regularly packed; along with annuli, membranous areas of various size and form are met in PMM. PMM are connected with the micronucleoli with filaments 20 nm thick. In the peripheral zone of the capsule, a variety of membranous structures is detected: intranuclear annuli lamellae, component of the capsule consists of different membranous and pseudomembranous (with annuli) structures. A question of the contribution of the chromatin material in the formation of the fibrous capsular component (PMM and membranous structures) is discussed.     

Genetic differentiation and speciation in Stomion (Goleoptera: Tenebrionidae): flightless beetles of the Galápagos Islands, Ecuador

Stomion is a swarm of 13 tenebrionid beetle taxa endemic to the Galápagos Islands. The taxa show marked morphological variation, and all are flighdess. They can be divided into three divergent morphological groups based on external sculpturing of the elytra. Eleven taxa were examined electrophoretically at 20 enzyme loci to measure the extent of genetic differentiation which accompanied morphological differentiation and speciation. Stomion are shown to be ideal candidates for allopatric and founder event speciation, with their limited dispersal ability, narrow distributions, and incidence of single-island endemics. Accordingly, individual heterozygosities were low in most taxa, and gene pool divergence was apparent among populations of single taxa occupying different islands. Both interspecific and intraspecific genetic identities were high, with an average 1 = 0.88 and I = 0.96, respectively. The relatively young age of the genus and the allopatric mode of speciation may account for high genetic relatedness. No clear relationship was found between allozyme phenotypes and the three morphological groups, thus, the evolution of these traits may have been facilitated by similar selection among islands of similar age and geography. The geographical distributions of the morphological groups can be used to suggest a general pattern of speciation for the group.     

The larval development of the telotrophic meroistic ovary in the bug Dysdercus intermedius (Heteroptera, Pyrrhocoridae)

Bug ovaries are of the telotrophic meroistic type. Nurse cells are restricted to the anterior tropharium and are in syncytial connection with the oocytes via the acellular trophic core region into which cytoplasmic projections of oocytes and nurse cells open. The origin of intercellular connections in bug ovaries is not well understood. In order to elucidate the cellular processes underlying the emergence of the syncytium, we analysed the development of the ovary of Dysdercus intermedius throughout the five larval instars. Up to the third instar, the germ cell population of an ovariole anlage forms a single, tight rosette. In the center of the rosette, phosphotyrosine containing proteins and f-actin accumulate. This center is filled with fusomal cytoplasm and closely interdigitating cell membranes known as the membrane labyrinth. With the molt to the fourth instar germ cells enhance their mitotic activity considerably. As a rule, germ cells divide asynchronously. Simultaneously, the membrane labyrinth expands and establishes a central column within the growing tropharium. In the fifth instar the membrane labyrinth retracts to an apical position, where it is maintained even in ovarioles of adult females. The former membrane labyrinth in middle and posterior regions of the tropharium is replaced by the central core to which nurse cells and oocytes are syncytially connected. Germ cells in the most anterior part of the tropharium, i.e. those in close proximity to the membrane labyrinth remain proliferative. The posterior-most germ cells enter meiosis and become oocytes. The majority of the ovarioles' germ cells, located in between these two populations, endopolyploidize and function as nurse cells. We conclude that the extensive multiplication of germ cells and their syncytial assembly during larval development is achieved by incomplete cytokineses followed by massive membrane production. Membranes are degraded as soon as the trophic core develops. For comparative reasons, we also undertook a cursory examination of early germ cell development in Dysdercus intermedius males. All results were compatible with the known basic patterns of early insect spermatogenesis. Germ cells run through mitotic and meiotic divisions in synchronous clusters emerging from incomplete cytokineses. During the division phase, the germ cells of an individual cluster are connected by a polyfusome rich in f-actin.