Abstract: Biocalcification is a widespread process of forming hard tissues like bone and teeth in vertebrates. It is also a topic connecting life sciences and earth sciences: calcified skeletons and shells deposited as sediments represent the earth's fossil record and are of paramount interest for biogeochemists trying to get an insight into the past of our planet. This study reports on the role of silicon in the early biocalcification steps, where silicon and calcium were detected on the surface of cyanobacteria (initial stage of lacustrine calcite precipitation) and in crustacean cuticles. By using innovative methodological approaches of correlative microscopy (AFM in combination with analytical TEM: EFTEM, EELS) the chemical form of silicon in biocalcifying matrices and organic-inorganic particles is determined. Previously, silicon was reported to be localized in active growth areas in the young bone of vertebrates. We have found evidence that biocalcification in evolutionarily distant organisms involves very similar initial phases with silicon as a key element at the organic-inorganic interface.
Abstract: A novel spotted fever group Rickettsia was found in Haemaphysalis sulcata ticks collected from sheep and goats in Croatia in 2006. At the same time, a genetically identical organism was co-isolated with the embryonic cell line CCE3 obtained from the soft tick Carios capensis in Georgia, USA. In this study, further phenotypic and genotypic characteristics of the novel rickettsial strain present in H. sulcata ticks were investigated. Based on the cultivation of bacteria in mosquito and Vero cell cultures, the presence of rickettsiae in tick tissues and cell cultures [confirmed by transmission electron microscopy (TEM)] and the amplification and sequencing of five rickettsial genes, it was demonstrated that the novel Rickettsia strain fulfils the criteria to be classified as a novel species. The name Rickettsia hoogstraalii sp. nov. is proposed for the new strain. Rickettsia hoogstraalii sp. nov., an obligately intracellular bacterium, was grown in Vero cells and arthropod CCE3, ISE6 and C6/36 cell lines. The morphology of the cells of the novel species was typical of SFG rickettsiae. The small coccobacillary appearance of the bacteria was apparent with light microscopy. A Gram-negative bacterial cell wall and a cytoplasmic membrane separated by a narrow periplasmic space were visible by TEM. To date, Rickettsia hoogstraalii sp. nov. has been isolated from two species of ticks, H. sulcata and C. capensis. The novel species appears to be geographically widely distributed, having been detected in Croatia, Spain and Georgia, USA. Although no information is available regarding the possible pathogenicity of the novel species for vertebrate hosts, R. hoogstraalii sp. nov. has a cytopathic effect in Vero, CCE3 and ISE6 cells. Sequence analyses of the 16S rRNA, 17 kDa, gitA, ompA and ompB genes indicated that even though R. hoogstraalii sp. nov. was closely related to Rickettsia fells, it represents a separate species within the spotted fever group. The type strain of R. hoogstraalii sp. nov. is strain Croatica(T) (=DSM 22243(T)=UTMB 00003(T)).
Abstract: The focused ion beam (FIB) and scanning electron microscope (SEM) are commonly used in material sciences for imaging and analysis of materials. Over the last decade, the combined FIB/SEM system has proven to be also applicable in the life sciences. We have examined the potential of the focused ion beam/scanning electron microscope system for the investigation of biological tissues of the model organism Porcellio scaber (Crustacea: Isopoda). Tissue from digestive glands was prepared as for conventional SEM or as for transmission electron microscopy (TEM). The samples were transferred into FIB/SEM for FIB milling and an imaging operation. FIB-milled regions were secondary electron imaged, back-scattered electron imaged, or energy dispersive X-ray (EDX) analyzed. Our results demonstrated that FIB/SEM enables simultaneous investigation of sample gross morphology, cell surface characteristics, and subsurface structures. The same FIB-exposed regions were analyzed by EDX to provide basic compositional data. When samples were prepared as for TEM, the information obtained with FIB/SEM is comparable, though at limited magnification, to that obtained from TEM. A combination of imaging, micro-manipulation, and compositional analysis appears of particular interest in the investigation of epithelial tissues, which are subjected to various endogenous and exogenous conditions affecting their structure and function. The FIB/SEM is a promising tool for an overall examination of epithelial tissue under normal, stressed, or pathological conditions.
Abstract: A novel spotted fever group Rickettsia was found in Haemaphysalis sulcata ticks collected from sheep and goats in Croatia in 2006. At the same time, a genetically identical organism was co-isolated with the embryonic cell line CCE3 obtained from the soft tick Carios capensis in Georgia, USA. In this study, further phenotypic and genotypic characteristics of the novel rickettsial strain present in H. sulcata ticks were investigated. Based on the cultivation of bacteria in mosquito and Vero cell cultures, the presence of rickettsiae in tick tissues and cell cultures [confirmed by transmission electron microscopy (TEM)] and the amplification and sequencing of five rickettsial genes, it was demonstrated that the novel Rickettsia strain fulfils the criteria to be classified as a novel species. The name Rickettsia hoogstraalii sp. nov. is proposed for the new strain. Rickettsia hoogstraalii sp. nov., an obligately intracellular bacterium, was grown in Vero cells and arthropod CCE3, ISE6 and C6/36 cell lines. The morphology of the cells of the novel species was typical of SFG rickettsiae. The small coccobacillary appearance of the bacteria was apparent with light microscopy. A Gram-negative bacterial cell wall and a cytoplasmic membrane separated by a narrow periplasmic space were visible by TEM. To date, Rickettsia hoogstraalii sp. nov. has been isolated from two species of ticks, H. sulcata and C. capensis. The novel species appears to be geographically widely distributed, having been detected in Croatia, Spain and Georgia, USA. Although no information is available regarding the possible pathogenicity of the novel species for vertebrate hosts, R. hoogstraalii sp. nov. has a cytopathic effect in Vero, CCE3 and ISE6 cells. Sequence analyses of the 16S rRNA, 17 kDa, gltA, ompA and ompB genes indicated that even though R. hoogstraalii sp. nov. was closely related to Rickettsia felis, it represents a separate species within the spotted fever group. The type strain of R. hoogstraalii sp. nov. is strain Croatica(T) (=DSM 22243(T)=UTMB 00003(T)).
Abstract: Because endogenous cellulases have been observed in arthropods, the potential ability to produce cellulose degrading enzymes was examined in the terrestrial isopod Porcellio scaber, an important decomposer of decayed plant material. cDNA fragments encoding portions of two novel endo-β-1,4-glucanase amino acid sequences were amplified by RT-PCR, and the amino acid sequences predicted were affiliated to endo-β-1,4-glucanases from other arthropods, where they cluster with endo-β-1,4-glucanases of decapod crustaceans. Hybridization in situ reveals the hepatopancreas to be the primary site of gene expression and provides direct evidence of the endogenous origin of endo-β-1,4-glucanase in P. scaber. Conservation of catalytically important amino acid residues suggests that both sequences translate into functional cellulases. Cellulolytic activity was detected in hepatopancreatic extract after separation by SDS-PAGE, which included CMC as substrate. This is the first evidence of endogenous cellulases in peracarid crustaceans and gives strong support for the involvement of isopod endo-β-1,4-glucanases in the degradation of cellulose in their diet.
Abstract: The focused ion beam (FIB) and scanning electron microscope (SEM) are commonly used in material sciences for imaging and analysis of materials. Over the last decade, the combined FIB/SEM system has proven to be also applicable in the life sciences. We have examined the potential of the focused ion beam/scanning electron microscope system for the investigation of biological tissues of the model organism Porcellio scaber (Crustacea: Isopoda). Tissue from digestive glands was prepared as for conventional SEM or as for transmission electron microscopy (TEM). The samples were transferred into FIB/SEM for FIB milling and an imaging operation. FIB-milled regions were secondary electron imaged, back-scattered electron imaged, or energy dispersive X-ray (EDX) analyzed. Our results demonstrated that FIB/SEM enables simultaneous investigation of sample gross morphology, cell surface characteristics, and subsurface structures. The same FIB-exposed regions were analyzed by EDX to provide basic compositional data. When samples were prepared as for TEM, the information obtained with FIB/SEM is comparable, though at limited magnification, to that obtained from TEM. A combination of imaging, micro-manipulation, and compositional analysis appears of particular interest in the investigation of epithelial tissues, which are subjected to various endogenous and exogenous conditions affecting their structure and function. The FIB/SEM is a promising tool for an overall examination of epithelial tissue under normal, stressed, or pathological conditions.
Abstract: Arthropods exhibit a vast variety of morphological forms. Developmental studies on diverse arthropod groups will help clarify how these forms evolved during arthropod evolution. The terrestrial isopod Porcellio scaber is a suitable novel animal model for developmental studies; it is well adapted to terrestrial habitats and these adaptations make the species interesting from the evolutionary perspective. Porcellio scaber is already extensively used in ecotoxicological and physiological studies and its biology is well known, but a comprehensive description of its ontogenetic development is still missing. Here, a staging system of P. scaber embryos and marsupial mancas based on morphological observations with light microscopy techniques is presented. An important adaptation of oniscids to terrestrial lifestyle is formation of specific structures in the digestive system, which enable effective water retention and good digestion of cellulose-rich plant material. Differentiation of the digestive system during P. scaber embryogenesis is described and the results show that digestive system is well developed already in late-stage embryos. The presented staging system should provide a useful basis for the expanding field of gene expression studies during arthropod embryogenesis and enable comparative developmental studies between arthropod groups.
Abstract: Rickettsia slovaca, a member of spotted fever rickettsiae, is an agent of a mild human disease known as Tibola or Debonel. Using quantitative real-time PCR we identified the highest point of multiplication of wild and standard type (strain B) of R. slovaca on the second vs. the fourth day postinfection. Comparing both types of R. slovaca by transmission electron microscopy substantiated different cytopathological and morphological changes in infected cells and other differences, for example a slight shift of stages during the life cycle that presented in a variety of forms and localization of the studied Rickettsia within the infected cells. Our study provides a valuable insight into the pathogenicity and virulence of R. slovaca.
Abstract: This study aimed at determining effects of Zn, Cd and their equitoxic mixtures on metal assimilation and food consumption of the terrestrial isopod Porcellio scaber, in relation to metal availability in the food. Cd was four times less water-extractable than Zn. Cd or Zn extractability was affected neither by metal concentration nor by the presence of the other metal. In single metal exposures, assimilation efficiency (AE) was up to five times higher for Cd than for Zn. In a mixture, AE of Cd significantly increased at low mixture concentrations and decreased at high mixture concentrations. AE of Zn significantly increased at intermediate mixture concentrations. Effects of the Zn and Cd mixture on food consumption were additive (28-day EC50,total = 1.10 TU; EC50,water-extractable = 1.18 TU) when based on total and water-extractable concentrations but antagonistic when related to internal metal concentrations in the isopods (EC50,internal = 1.40 TU). (C) 2009 Elsevier Inc. All rights reserved.
Abstract: Composition and spatial distribution of organic and inorganic materials within the cuticle of isopods vary between species. These variations are related to the behaviour and habitat of the animal. The troglobiotic isopod Titanethes albus lives in the complete darkness of caves in the Slovenian Karst. This habitat provides constant temperature and saturated humidity throughout the year and inconsistent food supply. These conditions should have lead to functional adaptations of arthropod cuticles. However, studies on structure and composition of cave arthropod cuticles are rare and lacking for terrestrial isopods. We therefore analysed the tergite cuticle of T albus using transmission and field-emission electron microscopy, confocal mu-Raman spectroscopic imaging, quantitative X-ray diffractometry, thermogravimetric analysis and atomic absorption spectroscopy. The ultrastructure of the epicuticle suggests a poor resistance against water loss. A weak interconnection between the organic and mineral phase within the endo- and exocuticle, a comparatively thin apical calcite layer, and almost lack of magnesium within the calcite crystal lattice suggest that the mechanical strength of the cuticle is low in the cave isopod. This may possibly be of advantage in maintaining high cuticle flexibility and reducing metabolic expenditures. (C) 2009 Elsevier Inc. All rights reserved.
Abstract: 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.
Abstract: This study aimed at determining effects of Zn, Cd and their equitoxic mixtures on metal assimilation and food consumption of the terrestrial isopod Porcellio scaber, in relation to metal availability in the food. Cd was four times less water-extractable than Zn. Cd or Zn extractability was affected neither by metal concentration nor by the presence of the other metal. In single metal exposures, assimilation efficiency (AE) was up to five times higher for Cd than for Zn. In a mixture, AE of Cd significantly increased at low mixture concentrations and decreased at high mixture concentrations. AE of Zn significantly increased at intermediate mixture concentrations. Effects of the Zn and Cd mixture on food consumption were additive (28-day EC(50,total)=1.10TU; EC(50,water-extractable)=1.18TU) when based on total and water-extractable concentrations but antagonistic when related to internal metal concentrations in the isopods (EC(50,internal)=1.40TU).
Abstract: Composition and spatial distribution of organic and inorganic materials within the cuticle of isopods vary between species. These variations are related to the behaviour and habitat of the animal. The troglobiotic isopod Titanethes albus lives in the complete darkness of caves in the Slovenian Karst. This habitat provides constant temperature and saturated humidity throughout the year and inconsistent food supply. These conditions should have lead to functional adaptations of arthropod cuticles. However, studies on structure and composition of cave arthropod cuticles are rare and lacking for terrestrial isopods. We therefore analysed the tergite cuticle of T. albus using transmission and field-emission electron microscopy, confocal micro-Raman spectroscopic imaging, quantitative X-ray diffractometry, thermogravimetric analysis and atomic absorption spectroscopy. The ultrastructure of the epicuticle suggests a poor resistance against water loss. A weak interconnection between the organic and mineral phase within the endo- and exocuticle, a comparatively thin apical calcite layer, and almost lack of magnesium within the calcite crystal lattice suggest that the mechanical strength of the cuticle is low in the cave isopod. This may possibly be of advantage in maintaining high cuticle flexibility and reducing metabolic expenditures.
Abstract: 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.
Abstract: Rickettsia slovaca, a member of spotted fever rickettsiae, is an agent of a mild human disease known as Tibola or Debonel. Using quantitative real-time PCR we identified the highest point of multiplication of wild and standard type (strain B) of R. slovaca on the second vs. the fourth day postinfection. Comparing both types of R. slovaca by transmission electron microscopy substantiated different cytopathological and morphological changes in infected cells and other differences, for example a slight shift of stages during the life cycle that presented in a variety of forms and localization of the studied Rickettsia within the infected cells. Our study provides a valuable insight into the pathogenicity and virulence of R. slovaca.
Abstract: We investigated the morphometric characteristics of the hepatopancreatic epithelium in the terrestrial isopod Porcellio scaber during acclimatization to laboratory conditions, during the daily cycle, the molt cycle, and fasting. The hepatopancreatic epithelium was analyzed using computer-assisted microscopy of serial sections of the hepatopancreatic tubes. In addition, the abundance, the distribution, and the size of lipid droplets in the hepatopancreatic epithelium were recorded. The experimental animals were collected in the field and transferred to the laboratory. The hepatopancreatic epithelium was thinner and lipid droplets reduced after 2 months of acclimatization to laboratory conditions. The daily cycle and the molt cycle affected neither the epithelial thickness nor the abundance of lipid droplets. But in animals fasted for 2 weeks, these two parameters were significantly reduced. Based on both the epithelial thickness and the abundance of lipid droplets in B cells, we propose criteria for estimating the stress status of the animals. With the possibility to determine the stress status, many studies on isopods gain in relevance.
Abstract: 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.
Abstract: 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 via] 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.
Abstract: Microscopic anatomy of the digestive system in embryos and larvae of the terrestrial isopod crustacean Porcellio scaber was investigated by light bright field, fluorescence and electron microscopy. During marsupial ontogenetic development the event-dependent staging was used to discriminate the various embryonic stages. At the late embryo stage the differentiation of the ectodermal part of the gut into the complex filtering foregut and the hindgut with absorptive and transporting functions is accomplished. The gut of the marsupial manca larva is fully developed and similar to that of the adult. In early embryos the endodermal midgut gland primordia are filled with yolk and lipid globules. In late embryos the epithelium of paired midgut gland tubes is composed of two cell types; one of them exhibits orange autofluorescence. The endodermal cells located between the foregut and the midgut glands of late embryos form the prospective midgut. The cells have electron dense cytoplasm, abundant glycogen fields, endoplasmic reticulum, dictyosomes and numerous vesicles. In the adults the endodermal cells of the midgut remain only in the midgut gland ducts which connect the midgut glands and the foregut. Details of the cellular ultrastructure and morphogenesis of the ectodermal and endodermal parts of the digestive system during embryonic development of Porcellio scaber provide data for further phylogenetic and comparative studies in peracaridan crustaceans and other arthropods. (c) 2008 Elsevier Ltd. All rights reserved.
Abstract: In Saccharomyces cerevisiae alcohol fermentation of 'Sauvignon blanc' grape must a low direct electric current (DC) of 1.3, 7.7 and 30 mu A was applied. Constant current stimulated wine yeasts metabolic activity by increasing production of glycerol and lactic acid was studied. The results of high performance liquid chromatography (HPLC) and gas chromatography (GC) indicated that by using the direct current at low temperature, similar results as those using higher fermentation temperatures can be achieved. Optical and transmission electron microscopy showed no visible morphological and ultra structural changes in cell morphology. The empirical experience resulting from present laboratory experiments offer a new approach in fermentation of grape musts wine and in wine process control.
Abstract: We report the use of a focused ion beam/scanning electron microscope (FIB/SEM) for simultaneous investigation of digestive gland epithelium gross morphology and ultrastructure of multilamellar intracellular structures. Digestive glands of a terrestrial isopod (Porcellio scaber, Isopoda, Crustacea) were examined by FIB/SEM and by transmission electron microscopy (TEM). The results obtained by FIB/SEM and by TEM are comparable and complementary. The FIB/SEM shows the same ultrastructural complexity of multilamellar intracellular structures as indicated by TEM. The term lamellar bodies was used for the multillamellar structures in the digestive glands of P. scaber due to their structural similarity to the lamellar bodies found in vertebrate lungs. Lamellar bodies in digestive glands of different animals vary in their abundance, and number as well as the thickness of concentric lamellae per lamellar body. FIB/SEM revealed a connection between digestive gland gross morphological features and the structure of lamellar bodies. Serial slicing and imaging of cells enables easy identification of the contact between a lamellar body and a lipid droplet. There are frequent reports of multilamellar intracellular structures in different vertebrate as well as invertebrate cells, but laminated cellular structures are still poorly known. The FIB/SEM can significantly contribute to the structural knowledge and is always recommended when a link between gross morphology and ultrastructure is investigated, especially when cells or cellular inclusions have a dynamic nature due to normal, stressed or pathological conditions.
Abstract: We investigated the morphometric characteristics of the hepatopancreatic epithelium in the terrestrial isopod Porcellio scaber during acclimatization to laboratory conditions, during the daily cycle, the Molt cycle and fasting. The hepatopancreatic epithelium was analyzed using computer-assisted microscopy of serial sections of the hepatopancreatic tubes. In addition, the abundance, the distribution. and the size of lipid droplets ill the hepatopancreatic epithelium were recorded. The experimental animals were collected ill the field and transferred to the laboratory. The hepatopancreatic, epithelium thinner and lipid droplets reduced after 2 months of acclimatization to laboratory conditions. The dally cycle and the molt cycle affected neither the epithelial thickness nor the abundance of lipid droplets. But in animals fasted Cor 2 weeks, these two parameters were significantly reduced. Based oil both the epithelial thickness and the abundance of lipid droplets in B cells, we propose criteria for estimating the stress status of the animals. With the possibility to determine the stress status, many Studies oil isopods gain in relevance. (C) 2008 Elsevier GmbH. All rights reserved.
Abstract: Microscopic anatomy of the digestive system in embryos and larvae of the terrestrial isopod crustacean Porcellio scaber was investigated by light bright field, fluorescence and electron microscopy. During marsupial ontogenetic development the event-dependent staging was used to discriminate the various embryonic stages. At the late embryo stage the differentiation of the ectodermal part of the gut into the complex filtering foregut and the hindgut with absorptive and transporting functions is accomplished. The gut of the marsupial manca larva is fully developed and similar to that of the adult. In early embryos the endodermal midgut gland primordia are filled with yolk and lipid globules. In late embryos the epithelium of paired midgut gland tubes is composed of two cell types; one of them exhibits orange autofluorescence. The endodermal cells located between the foregut and the midgut glands of late embryos form the prospective midgut. The cells have electron dense cytoplasm, abundant glycogen fields, endoplasmic reticulum, dictyosomes and numerous vesicles. In the adults the endodermal cells of the midgut remain only in the midgut gland ducts which connect the midgut glands and the foregut. Details of the cellular ultrastructure and morphogenesis of the ectodermal and endodermal parts of the digestive system during embryonic development of Porcellio scaber provide data for further phylogenetic and comparative studies in peracaridan crustaceans and other arthropods.
Abstract: We report the use of a focused ion beam/scanning electron microscope (FIB/SEM) for simultaneous investigation of digestive gland epithelium gross morphology and ultrastructure of multilamellar intracellular structures. Digestive glands of a terrestrial isopod (Porcellio scaber, Isopoda, Crustacea) were examined by FIB/SEM and by transmission electron microscopy (TEM). The results obtained by FIB/SEM and by TEM are comparable and complementary. The FIB/SEM shows the same ultrastructural complexity of multilamellar intracellular structures as indicated by TEM. The term lamellar bodies was used for the multillamellar structures in the digestive glands of P. scaber due to their structural similarity to the lamellar bodies found in vertebrate lungs. Lamellar bodies in digestive glands of different animals vary in their abundance, and number as well as the thickness of concentric lamellae per lamellar body. FIB/SEM revealed a connection between digestive gland gross morphological features and the structure of lamellar bodies. Serial slicing and imaging of cells enables easy identification of the contact between a lamellar body and a lipid droplet. There are frequent reports of multilamellar intracellular structures in different vertebrate as well as invertebrate cells, but laminated cellular structures are still poorly known. The FIB/SEM can significantly contribute to the structural knowledge and is always recommended when a link between gross morphology and ultrastrucutre is investigated, especially when cells or cellular inclusions have a dynamic nature due to normal, stressed or pathological conditions. (C) 2007 Published by Elsevier B.V.
Abstract: Pointed, rod-shaped bacteria colonizing the cuticular surface of the hindgut of the terrestrial isopod crustacean Porcellio scaber (Crustacea: Isopoda) were investigated by comparative 16S rRNA gene sequence analysis and electron microscopy. The results of phylogenetic analysis, and the absence of a cell wall, affiliated these bacteria with the class Mollicutes, within which they represent a novel and deeply branched lineage, sharing less than 82.6% sequence similarity to known Mollicutes. The lineage has been positioned as a sister group to the clade comprising the Spiroplasma group, the Mycoplasma pneumoniae group, and the Mycoplasma hominis group. The specific signature sequence was identified and used as a probe in in situ hybridization, which confirmed that the retrieved sequences originate from the attached rod-shaped bacteria from the hindgut of P. scaber and made it possible to detect these bacteria in their natural environment. Scanning and transmission electron microscopy revealed a spherically shaped structure at the tapered end of the rod-shaped bacteria, enabling their specific and exclusive attachment to the tip of the cuticular spines on the inner surface of the gut. Specific adaptation to the gut environment, as well as phylogenetic positioning, indicate the long-term association and probable coevolution of the bacteria and the host. Taking into account their pointed, rod-shaped morphology and their phylogenetic position, the name "Candidatus Bacilloplasma" has been proposed for this new lineage of bacteria specifically associated with the gut surface of P. scaber.
Abstract: Pointed, rod-shaped bacteria colonizing the cuticular surface of the hindgut of the terrestrial isopod crustacean Porcellio scaber (Crustacea: Isopoda) were investigated by comparative 16S rRNA gene sequence analysis and electron microscopy. The results of phylogenetic analysis, and the absence of a cell wall, affiliated these bacteria with the class Mollicutes, within which they represent a novel and deeply branched lineage, sharing less than 82.6% sequence similarity to known Mollicutes. The lineage has been positioned as a sister group to the clade comprising the Spiroplasma group, the Mycoplasma pneumoniae group, and the Mycoplasma hominis group. The specific signature sequence was identified and used as a probe in in situ hybridization, which confirmed that the retrieved sequences originate from the attached rod-shaped bacteria from the hindgut of P. scaber and made it possible to detect these bacteria in their natural environment. Scanning and transmission electron microscopy revealed a spherically shaped structure at the tapered end of the rod-shaped bacteria, enabling their specific and exclusive attachment to the tip of the cuticular spines on the inner surface of the gut. Specific adaptation to the gut environment, as well as phylogenetic positioning, indicate the long-term association and probable coevolution of the bacteria and the host. Taking into account their pointed, rod-shaped morphology and their phylogenetic position, the name "Candidatus Bacilloplasma" has been proposed for this new lineage of bacteria specifically associated with the gut surface of P. scaber.
Abstract: The objective of this study was to find out whether Porcellio scaber discriminates against Cd-contaminated food. The foraging behavior in animals offered uncontaminated and Cd-contaminated food simultaneously was quantified for 48-h employing computer-aided video tracking. To see whether the isopods' selection of less contaminated food could diminish the influence Cd on food consumption, growth, metal assimilation, moulting and mortality, Cd-dosed food (20, 45, 200 and 450 mg kg(-1) dry weight) was offered together with untreated food for 3 weeks. Data from the video tracking experiments revealed that animals visited Cd-dosed food as often as untreated food, but spent much less time near Cd-dosed food. Discrimination against Cd-contaminated food increased with previous experience with contaminated food and/or with increased Cd body burden. In 3 weeks exposure uncontaminated food preference rose with time of exposure and cadmium concentration in food and reached a maximal preference ratio of 65% (untreated food): 35% (Cd-dosed food). The decreased consumption of Cd-dosed food was compensated by the increased consumption of control food. Cadmium body burden increased with time of exposure and cadmium concentration in food consumed, while the influence of Cd on food consumption, growth and moulting was diminished.
Abstract: 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.
Abstract: GOAL, SCOPE AND BACKGROUND: Lake Skadar is the largest lake of the Balkan Peninsula, located along the Montenegro-Albanian border. The unique features of the lake and wide range of endemic, and rare or endangered plant and animal species, resulted in the classification of the Skadar as a wetland site of international significance. In spite of its importance, the lake is influenced by inflowing waters from the river Moraca and other regional rivers contaminated by the industry, municipal and agricultural activities in the area. Therefore, the lake has been the subject of various physical, chemical, biological and toxicological examinations. However, community-level analyses are most relevant to assess the effect of stressors on aquatic ecosystems. In the present study, bacterial community structure among differently polluted sites of the lake was compared using a genetic fingerprinting technique. METHODS: Water and sediment samples were collected from five differently polluted sampling sites on the Lake Skadar in spring and autumn of the same year. The bacterial community structure in the samples was characterized and compared by temporal temperature gel electrophoresis (TTGE) analysis of polymerase chain reaction-amplified bacterial 16S rRNA genes. RESULTS AND DISCUSSION: The TTGE analysis resulted in many distinguishable and reproducible band patterns, allowing a reliable comparison of bacterial communities among sampling sites. Results on the bacterial community structure revealed that three of the selected locations can be considered as sites that have not shown any pollution degradation determined by our method, due to similar structure of a bacterial community in the sediment samples. On the other hand, significant shifts in the bacterial community structure in the mouth of the rivers Moraca and Plavnica were shown. Since the results coincide with some of the bioassays and chemical analysis performed previously, the changes in bacterial community structure are explained as an effect of antropogenic pollution on the lake ecosystem by waters of the river Moraca and the stream Plavnica. CONCLUSION: The TTGE has proven to be an efficient and reliable method to monitor bacterial dynamics and community shifts in aquatic environments, especially in sediments. Within the variety of environmental quality assessments, the use of TTGE analyses from bacterial communities is strongly recommended, particularly as an initial investigation. However, in any conclusion on the state of the environment, the TTGE results should be combined to some other biological, chemical and hydrological data. RECOMMENDATION AND OUTLOOK: Since prokaryotes are a crucial group of organisms in the biosphere, the ecosystem function studies are largely based on bacterial communities. Therefore, bacterial community structure analysis should be a part of an integrated weight of evidence approach in pollution assessment. In case of Triad approach, consisting of chemical analyses, bioassays, and community studies in the field, the TTGE bacterial community structure analyses should be placed in the later Triad leg. In comparison to other community studies, based on various biotic indices, the TTGE bacterial community analysis has proven to be very sensitive, reliable and less time consuming.
Abstract: 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 mu g Zn/g dry food, 6 d) was applied to stimulate MT synthesis. After separation of the hindgut postmicrosomic 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 V-e/V(o)similar to 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.
Abstract: Goal, Scope and Background. Lake Skadar is the largest lake of the Balkan Peninsula, located along the Montenegro-Albanian border. The unique features of the lake and wide range of endemic, and rare or endangered plant and animal species, resulted in the classification of the Skadar as a wetland site of international significance. In spite of its importance, the lake is influenced by inflowing waters from the river Moraca and other regional rivers contaminated by the industry, Municipal and agricultural activities in the area. Therefore, the lake has been the Subject of various physical, chemical, biological and toxicological examinations. However, community-level analyses are most relevant to assess the effect of stressors on aquatic ecosystems. In the present study, bacteria] community structure among differently polluted sites of the lake was compared using a genetic fingerprinting technique. Methods. Water and sediment samples were collected from five differently polluted sampling sites on the Lake Skadar in spring and autumn of the same year. The bacterial community Structure in the samples was characterized and compared by temporal temperature gel electrophoresis (TTGE) analysis of polymerase chain reaction-amplified bacterial 16S rRNA genes. Results and Discussion. The TTGE analysis resulted in man), distinguishable and reproducible band patterns, allowing a reliable comparison of bacterial communities among sampling sites. Results on the bacterial community structure revealed that three of the selected locations call be considered as sites that have not shown any pollution degradation determined by Our method, due to similar structure of a bacterial community in the sediment samples. Oil the other hand, significant shifts in the bacterial community structure in the mouth of the rivers Mora a and Plavnica were shown. Since the results coincide with some of the bioassays and chemical analysis performed previously, the changes in bacterial community structure are explained as an effect of antropogenic pollution oil the lake ecosystem by waters of the river Moraca and the stream Plavnica. Conclusion. The TTGE has proven to be an efficient and reliable method to monitor bacterial dynamics and community shifts in aquatic environments, especially in sediments. Within the variety of environmental quality assessments, the use of TTGE analyses from bacterial communities is strongly recommended, particularly as an initial investigation. However, in any conclusion on the state of the environment, the TTGE results should be combined to some other biological, chemical and hydrological data. Recommendation and Outlook. Since prokaryotes are a crucial group of organisms in the biosphere, the ecosystem function Studies are largely based oil bacterial communities. Therefore, bacterial community structure analysis should be a part of an integrated weight of evidence approach in pollution assessment. In case of Triad approach, consisting of chemical analyses, bioassays, and community studies in the field, the TTGE bacterial community structure analyses should be placed in the later Triad leg. In comparison to other community studies, based on various biotic indices, the TTGE bacterial community analysis has proven to be very sensitive, reliable and less time consuming.
Abstract: In the mushrooms Pleurotus ostreatus and Agrocybe aegerita, expression of the hemolytic proteins ostreolysin and aegerolysin, which belong to the aegerolysin family, has been shown to be initiated specifically during formation of primordia and fruiting bodies. We used rabbit anti-ostreolysin and fluorescent rhodamine-labelled secondary goat antibodies for monitoring ostreolysin and aegerolysin in situ during the mushrooms' development. In parallel, the protein level in developing tissues was monitored with SDS-PAGE and hemolytic assay. Immunolocalization of ostreolysin, visualized by epifluorescence, together with biochemical tests, confirmed specific expression of ostreolysin and aegerolysin in the primordia and fruiting bodies but not in the vegetative mycelia. In the primordia, the proteins were disposed diffusely. In growing and mature fruit bodies they persisted in the lower part of the pileus, in particular in basidia and basidiospores, while in other parts only a few focal remains were observed. Confocal microscopy of immunolabelled sections showed that intracellular ostreolysin was located specifically along the inner edges of hyphae. Since both proteins were found preferentially in the rapidly growing primordia, and in the basidia and basidiospores of maturing fruit bodies, it is suggested that they might play a role in the processes of fructification and/or sporulation.
Abstract: The objective of this study was to find out whether Porcellio scaber discriminates against Cd-contaminated food. The foraging behavior in animals offered uncontaminated and Cd-contaminated food simultaneously was quantified for 48-h employing computer-aided video tracking. To see whether the isopods' selection of less contaminated food could diminish the influence Cd on food consumption, growth, metal assimilation, moulting and mortality, Cd-dosed food (20, 45, 200 and 450 mg kg(-1) dry weight) was offered together with untreated food for 3 weeks. Data from the video tracking experiments revealed that animals visited Cd-dosed food as often as untreated food, but spent much less time near Cd-dosed food. Discrimination against Cd-contaminated food increased with previous experience with contaminated food and/or with increased Cd body burden. In 3 weeks exposure uncontaminated food preference rose with time of exposure and cadmium concentration in food and reached a maximal preference ratio of 65% (untreated food): 35% (Cd-dosed food). The decreased consumption of Cd-dosed food was compensated by the increased consumption of control food. Cadmium body burden increased with time of exposure and cadmium concentration in food consumed, while the influence of Cd on food consumption, growth and moulting was diminished.
Abstract: In the mushrooms Pleurotus ostreatus and Agrocybe aegerita, expression of the hemolytic proteins ostreolysin and aegerolysin. which belong to the aegerolysin family, has been shown to be initiated specifically during Formation of primordia and fruiting bodies. We used rabbit anti-ostreolysin and fluorescent rhodamine-labelled secondary goat antibodies for monitoring ostreolysin and aegerolysin in situ during the. mushrooms' development. In parallel, the protein level in developing tissues was monitored with SDS-PAGE and hemolytic assay. Immunolocalization of ostreolysin, visualized by epifluorescence, together with biochemical tests, confirmed specific expression of ostreolysin and aegerolysin in the primordia and fruiting bodies but not in the vegetative mycelia. In the primordia, the proteins were disposed diffusely. In growing and Mature fruit bodies they persisted in the lower part of the pileus, ill particular in basidia and basidiospores, while in other parts only a few focal remains were observed. Confocal microscopy of immunolabelled sections showed that intracellular ostreolysin was located specifically along the inner edges of hyphae. Since both proteins were found preferentially in the rapidly growing primordia. an(] in the basidia and basidiospores of maturing fruit bodies, it is suggested that they might play a role in the processes of fructification and/or sporulation.
Abstract: 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. predominately oxic, conditions.
Abstract: Intracellular bacteria were observed in the hepatopancreas of the terrestrial isopod Porcellio scaber. Comparative 16S rRNA gene sequence analysis and electron microscopic observations were used to determine the taxonomic position of these intracellular bacteria. Phylogenetic analysis and a complex developmental cycle affiliate these bacteria to the order Chlamydiales, within which they form a distinctive lineage, close to the family Simkaniaceae. They share <92 % 16S rRNA gene sequence similarity with their closest relative and <88 % similarity with other members of the order Chlamydiales. A specific signature oligonucleotide sequence was identified and used as a probe, enabling the identification of intracellular bacteria in infected hepatopancreatic tissue. According to the distinctive morphology of their elementary bodies, which are rod-shaped rather than spherical and contain translucent oblong structures, their genomic properties and their crustacean host, the name 'Candidatus Rhabdochlamydia porcellionis' is proposed for intracellular bacteria in the hepatopancreas of P. scaber.
Abstract: The idea that terrestrial isopods regulate copper intake through the state of their copper stores by selection of an optimal copper diet has never been experimentally assessed. We investigated discrimination between untreated and Cu-enriched diets in the isopod Porcellio scaber in relation to their copper stores. Animals were pre-exposed to untreated or Cu-enriched poplar leaves. After 14 and 28 days of pre-exposure, two-way food choice experiments were performed for 14 days. Food consumption rates and the amount of copper accumulated due to the copper content of the food and the duration of pre-exposure were compared. Food selection and rejection as ways by which P. scaber may reduce or avoid the toxic effects of copper were evaluated. P. scaber offered both Cu-enriched food and untreated food preferred the latter, irrespective of the amount of assimilated copper. Copper body burdens increased with copper concentration in the food. In animals offered both Cu-enriched and untreated food, copper body burden also increased, but to a lower extent than in animals fed only a Cu-enriched diet. Therefore, food selection is a way of regulating copper intake, but only to a limited extent. The mortality of experimental animals increased significantly after longer exposure to highly Cu-enriched food. We conclude that in P scaber, the possibility of selecting food of different copper content may mitigate but cannot prevent the toxic effects of copper in copper-polluted environments. (C) 2003 Elsevier B.V. All rights reserved.
Abstract: The functional morphology of stomachs of the European semiterrestrial amphipod Orchestia cavimana and of the Australian terrestrial species Arcitalitrus sylvaticus was studied by electron microscopy. The stomach of the two amphipod species is divided longitudinally into a spacious dorsal food channel and two ventral filtration channels. Additionally, a prominent helically oriented circulation channel is situated on each lateral side of the stomach, forming a semicircular channel separated from the food channel by spines. The food channel conveys coarse food particles directly into the midgut through a funnel. The filtration channels receive fine material filtered through primary and secondary filters. Material forced through the secondary filters by the pressure of the laterally located inferolateralia eventually reaches the openings of the midgut glands. Washing of filters and soaking of ingested food items with enzymes probably is achieved by a forward stream of digestive juice from the midgut glands and conveyed through the circulatory channels. The specializations of the stomach of the two species of Amphipoda investigated are described and compared to the pertinent structures of Mysidacea and Isopoda.
Abstract: Intracellular bacteria were observed in the hepatopancreas of the terrestrial isopod Porcellio scaber. Comparative 16S rRNA gene sequence analysis and electron microscopic observations were used to determine the taxonomic position of these intracellular bacteria. Phylogenetic analysis and a complex developmental cycle affiliate these bacteria to the order Chlamydiales, within which they form a distinctive lineage, close to the family Simkaniaceae. They share <92% 16S rRNA gene sequence similarity with their closest relative and <88% similarity with other members of the order Chlamydiales. A specific signature oligonucleotide sequence was identified and used as a probe, enabling the identification of intracellular bacteria in infected hepatopancreatic tissue. According to the distinctive morphology of their elementary bodies, which are rod-shaped rather than spherical and contain translucent oblong structures, their genomic properties and their crustacean host, the name 'Candidatus Rhabdochlamydia porcellionis' is proposed for intracellular bacteria in the hepatopancreas of P. scaber.
Abstract: 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.
Abstract: The functional morphology of stomachs of the European semiterrestrial amphipod Orchestia cavimana and of the Australian terrestrial species Arcitalitrus sylvaticus was studied by electron microscopy. The stomach of the two amphipod species is divided longitudinally into a spacious dorsal food channel and two ventral filtration channels. Additionally, a prominent helically oriented circulation channel is situated on each lateral side of the stomach, forming a semicircular channel separated from the food channel by spines. The food channel conveys coarse food particles directly into the midgut through a funnel. The filtration channels receive fine material filtered through primary and secondary filters. Material forced through the secondary filters by the pressure of the laterally located inferolateralia eventually reaches the openings of the midgut glands. Washing of filters and soaking of ingested food items with enzymes probably is achieved by a forward stream of digestive juice from the midgut glands and conveyed through the circulatory channels. The specializations of the stomach of the two species of Amphipoda investigated are described and compared to the pertinent structures of Mysidacea and Isopoda.
Abstract: This paper describes a study of behavioural response in terrestrial isopod Oniscus asellus when offered two food pellets of different quality. One group had a choice of sterilised food and food pellets covered with mould. The other group had a choice of uncontaminated and cadmium-dosed food. During the behavioural test, the animals were monitored by a video camera and each visit to food pellets and time spent around it was counted. The results show that animals spent significantly less time near sterilised and cadmium-dosed food than with uncontaminated or mould-covered food. Discrimination between offered food pellets showed that avoidance behaviour can be used as an indicator of different food quality.
Abstract: 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.
Abstract: 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.
Abstract: Cellular ultrastructure varies in accordance with physiological processes, also reflecting responses to environmental stress factors. Ultrastructural changes of the hepatopancreatic cells in the terrestrial isopod Porcellio, scaber exposed to sublethal concentrations of zinc or cadmium in their food were identified by transmission electron microscopy. The exclusive structural characteristic of the hepatopancreas of animals exposed to metal-dosed food was grain-like electrondense deposits (EDD) observed in the intercellular spaces and in vesicles of B cells. In addition, hepatopancreatic cells of metal-exposed animals displayed non-specific, stress-indicating alterations such as cellular disintegration, the reduction of energetic reserves (lipid droplets, glycogen), electron dense cytoplasm, ultrastructural alterations of granular endoplasmic reticulum (GER), the Golgi complex and mitochondria. (C) 2002 Elsevier Science B.V. All rights reserved.
Abstract: Terrestrial isopods Porcellio scaber (Crustacea) were fed for five weeks on food contaminated by 250, 500, or 1,000 mug of Cd/g or for 10 d on diets with 50 or 250 mug Cd/g food. In both experiments, fecal production rate and colony forming units (CFUs) in the guts were determined. In addition, at the end of 10 d, each distinct colony morphotype obtained in gut samples was purified and characterized. Isolates were separated into 25 groups based on morphological and biochemical characteristics. These bacterial groups were used as units for calculating Shannon equitability indices (J) for each gut. The relative frequencies of the 25 bacterial units were determined in both cadmium groups (50 or 250 mug Cd/g food) and in the control. Cadmium-induced perturbations observed in the gut microbial communities were (1) increased number of morphologically distinct bacterial isolates in the group fed low-cadmium-dosed food (50 mug Cd/g) and reduced number of morphologically distinct bacterial isolates in the group fed high-cadmium-dosed food (250 mug Cd/g) compared with the control, (2) increased or decreased relative frequencies of almost all 25 bacterial units provoked by cadmium-contaminated food, (3) time-dependent increased numbers of gut CFUs in cadmium-fed animals (dose dependence was not observed), and (4) significant changes in community structure described by Shannon equitability indices at lower levels of food contamination (50 mug Cd/g) only. Gut microflora parameters are proposed as additional endpoints in the standardized single-species toxicity test with the terrestrial isopod P. scaber as a means of increasing the ecological relevance of the results.
Abstract: Molecular approaches were used to examine the genetic diversity of bacteria associated with the gut wall of the terrestrial isopod Porcellio scaber and to determine whether an autochthonous microflora exists in the P. scaber hindgut. 16S ribosomal genes were amplified from the total DNA isolated from thoroughly washed papillate regions of the hindgut, where the highest concentrations of bacteria are commonly found. The amplified genes were cloned, sequenced and phylogenetically analysed. The results implied an unexpectedly large diversity of microflora associated with the cuticle of the hindgut. Almost half of the retrieved sequences were found to be less than 80% homologous with any of the known sequences available at DNA data banks. Most of these sequences were clustered in one of three groups, and were clearly distant from the sequences of other bacterial taxa, indicating that they could represent novel bacterial species or even genera. More than two thirds of the sequences were found to be phylogenetically related to sequences from bacteria typically isolated from human and animal intestines, e.g. streptococci, enterococci, and members of the genus Bacteroides. The majority of the remaining sequences were most closely related to typical soil bacteria, e.g. bacilli and pseudomonads. The facts that a large proportion of the retrieved sequences was related to the sequences of bacteria, which are autochthonous to intestinal ecosystems, and that bacteria, specifically attached to the cuticular spines, were observed, indicate that truly autochthonous bacteria may well be present in the hindgut of P. scaber. (C) 2002 Published by Elsevier Science B.V. on behalf of the Federation of European Microbiological Societies.
Abstract: The scavenging cirolanid isopod Natatolana obrusata was caught using primarily meat-baited traps at a station in the Weddell Sea at Kap Norvegica. The hindguts of most specimens were densely stuffed with meat from the traps. The anatomy and the ultrastructure of different sections of the digestive system are described. The large and extremely expandable mouth opens into a short oesophagus which extends posteriorly into a small stomach. Lateralia and primary filter can be observed directly when the mouth opening is widened to its extreme. The anterior hindgut is extremely variable in its lumen and very distensible, depending on the nutritional condition. The cuticle of the hindgut is colonized by fungi. The length of the midgut glands varies with the nutritional conditions. Morphology and anatomy of the digestive system of Natatolana obtusata are discussed with the background of the lifestyle of the species and compared with other taxa.
Abstract: Terrestrial isopods Porcellio scaber (Crustacea) were fed for five weeks on food contaminated by 250, 500, or 1,000 microg of Cd/g or for 10 d on diets with 50 or 250 microg Cd/g food. In both experiments, fecal production rate and colony forming units (CFUs) in the guts were determined. In addition, at the end of 10 d, each distinct colony morphotype obtained in gut samples was purified and characterized. Isolates were separated into 25 groups based on morphological and biochemical characteristics. These bacterial groups were used as units for calculating Shannon equitability indices (J) for each gut. The relative frequencies of the 25 bacterial units were determined in both cadmium groups (50 or 250 microg Cd/g food) and in the control. Cadmium-induced perturbations observed in the gut microbial communities were (1) increased number of morphologically distinct bacterial isolates in the group fed low-cadmium-dosed food (50 microg Cd/g) and reduced number of morphologically distinct bacterial isolates in the group fed high-cadmium-dosed food (250 microg Cd/g) compared with the control, (2) increased or decreased relative frequencies of almost all 25 bacterial units provoked by cadmium-contaminated food, (3) time-dependent increased numbers of gut CFUs in cadmium-fed animals (dose dependence was not observed), and (4) significant changes in community structure described by Shannon equitability indices at lower levels of food contamination (50 microg Cd/g) only. Gut microflora parameters are proposed as additional endpoints in the standardized single-species toxicity test with the terrestrial isopod P. scaber as a means of increasing the ecological relevance of the results.
Abstract: Molecular approaches were used to examine the genetic diversity of bacteria associated with the gut wall of the terrestrial isopod Porcellio scaber and to determine whether an autochthonous microflora exists in the P. scaber hindgut. 16S ribosomal genes were amplified from the total DNA isolated from thoroughly washed papillate regions of the hindgut, where the highest concentrations of bacteria are commonly found. The amplified genes were cloned, sequenced and phylogenetically analysed. The results implied an unexpectedly large diversity of microflora associated with the cuticle of the hindgut. Almost half of the retrieved sequences were found to be less than 80% homologous with any of the known sequences available at DNA data banks. Most of these sequences were clustered in one of three groups, and were clearly distant from the sequences of other bacterial taxa, indicating that they could represent novel bacterial species or even genera. More than two thirds of the sequences were found to be phylogenetically related to sequences from bacteria typically isolated from human and animal intestines, e.g. streptococci, enterococci, and members of the genus Bacteroides. The majority of the remaining sequences were most closely related to typical soil bacteria, e.g. bacilli and pseudomonads. The facts that a large proportion of the retrieved sequences was related to the sequences of bacteria, which are autochthonous to intestinal ecosystems, and that bacteria, specifically attached to the cuticular spines, were observed, indicate that truly autochthonous bacteria may well be present in the hindgut of P. scaber.
Abstract: 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 mu 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 mu m were filled predominately with spherical bacteria but rod-shaped forms were also present in large numbers. Larger vacuoles of 10 to 20 mu 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, (C) 1999 Academic Press.
Abstract: 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.
Abstract: The hepatopancreata of seven mature females of the shrimp Palaemon elegans from a rock pool in the Bay of Piran, Adriatic Sea, were examined for pathogens. Five of the seven females investigated were infected with four different pathogens, two viruses and two bacteria. One specimen was infected with Palaemon B-cell Reo-like Virus (PBRV), one with Bay of Piran Shrimp Virus (BPSV), one with Hepatopancreatic Brush border Lysis-bacteria (HBL), one with rickettsiae and one with both PBRV and rickettsiae. PBRV, BPSV and the HBL-bacteria turned out to be new shrimp pathogens. All of the four pathogens induced agent-specific transformations in the infected hepatopancreas cells. Although the viruses and bacteria had not seriously affected the vitality of the specimens investigated they may cause problems under less favourable nutritional and environmental conditions.
Abstract: The moult cycle of Porcellio scaber was studied in order to determine the typical shape of sternal calcium deposits during premoult, Males and non-breeding females were observed for 13 weeks. According to the appearance, size, and shape of sternal calcium deposits, premoult was divided into three substages which occurred in all animals investigated. The easiest and the least disturbing way to determine the moult stage of isopods in routine experiments is to identify one of the premoult substages. The duration of the moult cycle in both sexes of Porcellio scaber is approximately 33 days, with greater variation in females. The proposed procedure for moult stage determination is simple and rapid.
Abstract: 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.
Abstract: The effect of zinc-contaminated leaf litter (250-10,000 mu g/g dry wt.) diets on molting in Porcellio scaber, a terrestrial isopod was measured under controlled conditions. The duration of the premolt stage, the period between two successive ecdyses and the rare of molting, was followed during the exposure. Increased zinc concentration in the food caused prolongation of the molt cycle and decreased molt frequency. The duration of the premolt stage was not changed. Molt cycle was not affected drastically due to food deprivation. The application of molt cycle response to contaminated food in toxicity tests is discussed.
Abstract: 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.
Abstract: The cave-dwelling shrimp, Troglocaris schmidtii, has a unique mode of lipid storage. The lipid lies extracellularly in specialized compartments of the hepatopancreas, named oleospheres. The lipid is synthesized in the R-cells of the hepatopancreatic epithelium and accumulates in lipid droplets which fuse to form bigger globules. Mature lipid globules display moderately electron dense centers probably comprising triglycerides, and a broad electron dense boundary presumably consisting of lipoproteins. The globules are discharged into the lumen of the hepatopancreatic tubules by a kind of apocrine secretion. There, they coalesce to form larger masses. Finally, these lipid masses are transported into the oleospheres through a valve-like structure. The continual accumulation of lipid results in a drastic expansion of the oleospheres up to 500-mu-m in diameter. The absence of food in the digestive tract and the inactivity of the digestive enzyme producing F-cells indicate that digestion is suspended in the period of oleosphere formation. The curious mode of lipid storage in T. schmidtii may represent an adaptation to the extreme environmental conditions of a cave.
Abstract: The investigation of the foregut-hindgut junction with scanning and transmission electron microscope demonstrated the presence of midgut cells in the digestive tract of Ligia italica. The ultrastructural features of the cells suggest that they are involved in absorption and secretion.
