Metal contamination recorded in the sediment of the semi-closed Bakar Bay (Croatia).

This study presents metal levels in the sediments of the Bakar Bay, with its main goal to evaluate recent anthropogenic influence, as well as over previous decades. Sediment profiles at 7 sampling points were taken. Chemical contents in bulk sediment were obtained using ICP, ICP-MS, and AAS methodologies, and 20 most significant elements were presented. Concentrations of selected elements were evaluated by factor statistical analyses to identify their source. Also, metal enrichment factor and geoaccumulation index were calculated, and spatial distribution maps for three sediment layers were constructed. Measured metal concentrations in sediment were compared with concentrations in other sediments from the Adriatic Sea. In addition, a set of sediment quality guidelines were also applied in order to predict the probability of adverse biological effects on the benthic community: This was found not to be very serious. Factor analysis clearly demonstrates the segregation between metals of natural origin resulted from soil and bedrock weathering (Li, Al, Cr, Sc), and with two anthropogenic sources originating from the city of Bakar and bulk cargo terminal (Hg, Pb, Zn, Ag, Sn, and Fe). Mercury (max 0.65 µg g(-1)) is found to be the heaviest contaminant, followed by lead (max 71.5 µg g(-1)), copper (89.3 µg g(-1)), and zinc (156 µg g(-1)). However, this study shows that Bakar Bay is considerably less polluted with toxic metals than it was believed.

An assessment of mercury loading in core sediments of Sunderban mangrove wetland, India (a preliminary report).

This is a preliminary report on total mercury (T(Hg)) in core sediments (<63 microm particle size) of Sunderban mangrove wetland, northeastern part of the Bay of Bengal, India. Cold vapor atomic absorption spectrometry (CVAAS) was used for T(Hg) determination. The concentration varies from 9.8 to 535.1 ppb (ngg(-1)). Results revealed variations over premonsoon and postmonsoon month at different core depth, as well as in studied three sampling stations, located at the site of three rivers: Hugli River (S(1)), Matla River (S(2)) and Bidyadhari River (S(3)). Elevated concentration of T(Hg) in subsurface layer (4-8 cm) of the core at S(2) is attributed to remobilization of mercury from deeper sediment (32-36 cm). Positive correlation is present between total Hg and clay content. Based on index of geoaccumulation (I(geo)) and Effects-Range Low (ER-L) value (150 ppb) it is considered that the sediments are till now unpolluted. As a consequence, there is less chance of ecotoxicological risk to organisms living in studied sediments. Two statistical methods were applied to determine T(Hg) anomalies. Box plot method showed one extreme and three outliers in S(1) at postmonsoon season. Two extremes were found at S(2) at 4-8 and at 32-36 cm in premonsoon period. In S(3) there was no anomaly by box plot method. MAD method was more sensitive than box plot method and T(Hg) anomaly was detected at 12-16 cm in S(3) during postmonsoon season. The data reported are useful baselines for T(Hg) in Sunderban mangrove wetland, India and would be of help in future sediment quality studies.

Multidisciplinary work on barium contamination of the karstic upper Kupa River drainage basin (Croatia and Slovenia); calling for watershed management.

The present work was designed as an extension of a previous study of a barium anomaly observed in stream sediments of the Kupa River. In its upper part the Kupa River drains a region underlain by a trans-boundary aquifer. The river is a significant water resource in a region of tourism, sport, and fishing in both Croatia and Slovenia. The contamination source is situated in Homer (Lokve), Croatia, where barite was mined until 10 years ago. The barium processing waste material (<3-mm fraction) was carelessly deposited in gardens, forests, and into a sinkhole, which has an underground link with the Kupica River, a tributary of the Kupa River. Barium waste and stream sediments were analyzed using comparative techniques: X-ray diffraction (XRD), X-ray fluorescence (XRF), Mössbauer spectroscopy, and grain size analysis. XRD of the waste material identified the major minerals quartz, barite, and dolomite and the Fe-containing minor minerals muscovite and goethite. Barite was identified as a minor or trace mineral in the Kupica River sediments. XRF analysis of the waste material has shown Ba and Fe to be the predominant elements, Ca and K to be minor elements, and Mn, Zn, Sr, Pb, Co, Cu, As, Zr, Rb, Y, and Mo to be trace elements. Mössbauer spectroscopy performed at room temperature (RT) was used to study iron minerals, particularly to obtain information on the valence status of Fe ions. Grain size analysis of the waste material (<63-microm fraction) has shown that it contains 23.5% clay-size material in comparison with 7-8% clay-size material in stream sediments. It is our aim to combine geochemical and medical methods to investigate the possible impact of waste disposal on human health in Lokve. At this stage of the work, concentrations of Ba and other toxic elements in the water compartment of the Kupica River (a source of drinking water) have not been monitored by Croatian Waters (name of the Croatian water authorities). The necessity of such measurements in future studies has been highlighted. A preliminary study of diseases diagnosed in Lokve shows that about 18% of the total inhabitants have serious medical problems. Diseases of the circulatory system, endocrine, nutritional, and metabolic diseases, neoplasms, and respiratory diseases predominate. This paper calls for further multidisciplinary research on the health effects of barium and trace elements, as well as for bioremediation of contaminated gardens and for watershed management of vulnerable karstic aquifers.

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.

Mitochondrial ribosomal RNA in the germinal granules in Xenopus embryos revisited.

Germ cells of various animals contain a determinant that is called the germ plasm. In amphibians such as Xenopus laevis, the germ plasm is composed of mitochondria and electron dense germinal granules that are embedded in a fibrillar matrix. Previous reports indicated that one of the components of germinal granules was mitochondrial large and small ribosomal RNA (mtlrRNA and mtsrRNA). Utilizing a modified procedure for electron microscopy in situ hybridization, we investigated the distribution of these RNAs along with other components of the germ plasm in Xenopus laevis embryos. We found, that contrary to previous reports, the mtlrRNA and mtsrRNA were located in close vicinity to the germinal granules but were not major constituents of granules. The majority of the mtlrRNA and mtlsrRNAs was present inside the mitochondria and in the germ plasm matrix.

The vegetally localized mRNA fatvg is associated with the germ plasm in the early embryo and is later expressed in the fat body.

Vegetally localized RNAs in Xenopus oocytes have been implicated in the establishment of the primary germ layers and the formation and development of the primordial germ cells. fatvg mRNA is localized through the late pathway to the vegetal cortex. Like Vg1 mRNA fatvg is distributed throughout the entire cortex; however, unlike Vg1 there is a small fraction of the fatvg mRNA that is associated with the mitochondrial cloud. In early cleavage stage embryos, fatvg mRNA is associated with the germ plasm located at the tips of the vegetal blastomeres of the embryo. While several localized RNAs that follow the Message Transport Organizer (METRO) pathway have been found in the germ plasm in embryos, fatvg is a late pathway RNA that is associated with the germ plasm. In tadpoles, fatvg mRNA shows a novel pattern of expression which is distinct from the germ cell lineage and is detected at the dorso-anterior margin of the endodermal mass along the midline in two clusters of cells. fatvg mRNA expression is also detected later in the developing fat bodies, the major adipose tissues of the frog.

RNA localization and germ cell determination in Xenopus.

In many organisms the proper development of the embryo depends on the asymmetrical distribution of maternal RNAs and proteins in the egg. Although the Xenopus oocyte is radially symmetrical it contains distinct populations of maternal RNAs that are localized either in the animal or vegetal pole. The process of localization of RNAs in Xenopus oocytes occurs during the long period of oocyte differentiation and growth that is accompanied by the elaboration of oocyte polarity. Some of the vegetally localized RNAs, such as Vg1, VegT, and Xwnt11, are involved in axial patterning and germ layer specification. Others, such as Xdazl and Xcat2, which are located in the germ plasm, are likely to play a role in the specification of germ cell fate. We will discuss the different aspects of RNA localization in Xenopus in the context of the differentiation of the germ cells and the development of the oocyte polarity.

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).

The targeting of Xcat2 mRNA to the germinal granules depends on a cis-acting germinal granule localization element within the 3'UTR.

The germ cell lineage is specified by the germ plasm, which in Xenopus laevis contains putative determinants called germinal granules. The pathway through which these structures form and how their components are assembled remain unclear. Using a combination of electron microscopy and in situ hybridization with the germinal granule-associated Xcat2 mRNA we demonstrated that the granules were derived from a branching network of granulofibrillar material within the mitochondrial cloud. Targeting of Xcat2 mRNA to the germinal granules depended on a 164-nt 3'UTR germinal granule localization element (GGLE; nt 631-795) that was distinct from the previously defined mitochondrial cloud localization element (MCLE; nt 403-630; Y. Zhou and M. L. King, 1996, Development 122, 2947-2953). This demonstrated that the Xcat 3'UTR contains a compound localization element consisting of a general element (MCLE) targeting the RNA to the mitochondrial cloud and a second element (GGLE) responsible for targeting to the germinal granules within the cloud. The GGLE when fused to Xlsirt RNA was sufficient to target this nongranule mitochondrial cloud-associated RNA to the germinal granules. This is the first example of a localization element involved in targeting an mRNA to a specific subcellular target such as the germinal granules and suggests that cis-acting elements on RNAs play an important role in the assembly of germinal granules and, therefore, the establishment of the germ cell lineage.

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.