Ultrastructural study of the life cycle of Rickettsia slovaca, wild and standard type, cultivated in L929 and Vero cell lines.

Ultrastructural changes induced by Rickettsia slovaca standard type (ST) and wild type (WT) were examined during their life cycle in L929 and Vero cells. R. slovaca invaded the cytoplasm of the host cell by phagocytosis on the 1st d p.i. Rickettsiae adhering to the cytoplasmic membrane were engulfed by cellular extensions and occurred in phagocytic vacuoles. Binary fission of rickettsia was observed. The nuclear chromatin of eukaryotic cells was rearranged and condensed during 3rd and 6th d p.i. Finally, loss of the plasma membrane integrity, destruction of cytoplasm and nucleus resulted in cell lysis. Degeneration of the host cell caused by WT and ST was observed after 4 and 5 d p.i. in L929 cells and after 3 and 6 d p.i. in Vero cells, respectively. WT type was able to penetrate into the nucleus of the host cell and was responsible for dilatation of the perinuclear space and endoplasmic reticulum, causing more pronounced and different cytopathological changes than the ST.

Rickettsial agents in Slovakian ticks (Acarina, Ixodidae) and their ability to grow in Vero and L929 cell lines.

A total of 80 adult ticks (55 Haemaphysalis inermis, 12 Dermacentor reticulatus, 11 D. marginatus, 2 Ixodes ricinus) were collected from vegetation in three areas of Slovakia (forest and pasture habitat) in central Europe. Forty-six (46 ticks) (57.5%) of all species tested were positive by the hemocyte test, PCR assays based on the gltA and ompA genes showed a Rickettsiaceae infection in 77.5% of the ticks, whereas only one H. inermis tick was positive for Anaplasmataceae on a 16S rRNA-based PCR. Isolation of rickettsiae was attempted on all collected ticks by means of the shell vial technique, 52 isolates of which were inoculated into Vero cells and 28 into L929 cells. Rickettsiae were detected in 50% (40/80) of the cell lines using the Gimenez staining method, whereas 33.8% (27/80) of the cell lines were PCR-positive for Rickettsia species. The presence of rickettsiae was shown by PCR to be around 30.8% (16/52) in Vero and 39.3% (11/28) in L929 cell lines. Sequencing results showed that detected infections were Rickettsia sp., R. raoultii, and Anaplasma phagocytophilum in ticks, and R. slovaca in cell lines. This is the first report of R. raoultii in Slovakia. Observations by electron microscopy of the R. slovaca isolate from Vero cell lines showed a microcapsular layer, typical Gram-negative cell wall, and a cytoplasmic membrane.

Metallothionein-like proteins and zinc--copper interaction in the hindgut of Porcellio scaber (Crustacea: Isopoda) exposed to zinc.

Metallothioneins (MTs) are ubiquitous low-molecular-weight metal-binding proteins, with a variety of functions in metal metabolism ascribed to them. Among terrestrial invertebrates, MTs have been studied in nematodes, insects, snails, and earthworms. The aim of this study was the characterization of MT-like proteins in the terrestrial isopod crustacean Porcellio scaber in order to analyze their probable role in the metabolism of copper (Cu) and zinc (Zn). Dietary Zn supplementation (793 microg Zn/g dry food, 6 d) was applied to stimulate MT synthesis. After separation of the hindgut post-microsomic supernatant (cytosol) of Zn-exposed animals by gel filtration on a Sephadex G-75 column, a Cu- and Zn-containing peak was detected in the position of Ve/Vo approximately 2, where MTs are expected to elute. Rechromatography of these fractions by size-exclusion chromatography-high-performance liquid chromatography revealed that the 215-nm absorbance peak coincided with the absorbance peak of the rabbit MT II standard. These low-molecular-weight Cu- and Zn-binding compounds, detected in the cytosol of the hindgut cells in Zn-exposed P. scaber, are considered to be Cu, Zn-MT-like proteins. To our knowledge, this is the first report on the characterization of MT-like proteins in isopod crustaceans. These results also indicate that both Zn and Cu dynamics in P. scaber hindgut are affected at the given dietary Zn supplementation and that MT-like proteins are involved in this Zn-Cu interaction.

Anaerobic bacteria in the gut of terrestrial isopod Crustacean Porcellio scaber.

Anaerobic bacteria from Porcellio scaber hindgut were identified and, subsequently, isolated using molecular approach. Phylogenetic affiliation of bacteria associated with the hindgut wall was determined by analysis of bacterial 16S rRNA gene sequences which were retrieved directly from washed hindguts of P. scaber. Sequences from bacteria related to obligate anaerobic bacteria from genera Bacteroides and Enterococcus were retrieved, as well as sequences from 'A1 subcluster' of the wall-less mollicutes. Bacteria from the genus Desulfotomaculum were isolated from gut wall and cultivated under anaerobic conditions. In contrast to previous reports which suggested the absence of anaerobic bacteria in the isopod digestive system due to short retention time of the food in the tube-like hindgut, frequent renewal of the gut cuticle during the moulting process, and unsuccessful attempts to isolate anaerobic bacteria from this environment our results indicate the presence of resident anaerobic bacteria in the gut of P. scaber, in spite of apparently unsuitable, i.e. predominantly oxic, conditions.

A contribution to the functional morphology of the femoral chordotonal organ in the green lacewing Chrysoperla carnea (Neuroptera).

The femoral chordotonal organ (FCO) and the subgenual organ (SGO) of the green lacewing Chrysoperla carnea were examined by conventional light and confocal laser scanning microscopy in order to search for neuroactive substances which are used for neurotransmission in sensory cells of these organs. Antibodies against serotonin, histamine and choline acetyltransferase were tested immunohistochemically. In the FCO, antiserum against serotonin strongly labelled cell bodies and axons of about 16 sensory cells. In the proximal scoloparium all 12 sensory cells showed immunoreaction with antiserotonin. In the distal scoloparium only four of 40 sensory cells were immunoreactive. These results suggest that different neuroactive substances are employed as neurotransmitters in the FCO of the green lacewing and that the proximal scoloparium and the distal scoloparium are functionally differentiated. Contrary to the FCO in the locust, acetylcholine was not found as a neurotransmitter in the FCO of the green lacewing. Additionally, histamine showed a negative result in the sensory cells of the FCO. Other neuroactive substances seem to be used as transmitters in the SGO because none of the tested antibodies showed positive reaction.

Morphological description of bacterial infection of digestive glands in the terrestrial isopod porcellio scaber (Isopoda, crustacea)

Morphological studies of the hepatopancreas of the terrestrial isopod Porcellio scaber revealed bacterial infection. The percentage of infected animals collected from the same site varied from 0 to 10% during the 4 years of study. Transmission electron microscopy, scanning electron microscopy, and light microscopy revealed that infected glands differed from those in healthy isopods. The most prominent sign was white spots between 100 and 200 &mgr;m in diameter along the entire gland. These spots were aggregations of vacuoles in the cells that were densely filled with bacteria in different phases of the developmental cycle that included the formation of small, dense, rod-shaped infective bacteria and much larger spherical multiplying cells filled with aggregates of polysomes and a chromatin network. Occasionally, large sphericles were filled with homogeneous electron-dense material. Bacteria were not observed in the cell nucleus. Small vacuoles of less than 5 &mgr;m were filled predominately with spherical bacteria but rod-shaped forms were also present in large numbers. Larger vacuoles of 10 to 20 &mgr;m in the main were densely filled with rod-shaped bacteria. According to the literature on the morphological characteristics of bacteria infecting invertebrates, those described in our study would be classified in the genus Rickettsiella. However, most recent investigations show that besides morphological investigations, genetic ones are also needed to define the taxonomic position of bacteria that infect invertebrates. Copyright 1999 Academic Press.

The effect of Zn on the digestive gland epithelium of Porcellio scaber (Isopoda, Crustacea).

The effect of zinc (Zn)-contaminated food on the shape of digestive gland epithelium was studied in the terrestrial isopod, Porcellio scaber. In animals fed with 5,000 micrograms Zn g-1 dry wt. of food, the epithelium was flattened in the anterior part of the gland tube. In the posterior part of the gland tube, the shape and size of cells did not change drastically, but they had folded apical surface. In animals fed with more Zn-contaminated food (10,000 micrograms Zn g-1 dry wt.) the epithelium was uniformly flattened and the basal lamina was intensively folded. In both cases, reduction of lipid bodies was evident. The possibility of using the shape of the gland epithelium as a biomarker of toxic chemicals is discussed.

Ultrastructural analysis of the integument during the moult cycle in Ligia italica (Crustacea, Isopoda).

The formation of the cuticle was investigated during moulting of the isopod crustacean Ligia italica. The intermoult cuticle is a four-layered lamellar structure composed of chitin-protein fibrils and mineralized in its upper half. The distribution of calcium carbonate in cuticle during moult cycle was determined by cytochemical methods and X-ray microanalysis, Epi-and exocuticle are secreted during premoult. Calcium is resorbed from the old cuticle and accumulates in the ecdysal gap as calcium granules. Endocuticle is secreted after moult when the mineralization of exocuticle starts. The shape and ultrastructure of epithelial cells change during cuticle secretion and mineralization. Mitochondria, bundles of filaments, calcium granules and large amounts of glycogen accumulate in the apical cytoplasm of cells in premoult animals.