Jasna Štrus

Rickettsia hoogstraalii sp. nov., isolated from hard- and soft-bodied ticks

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)).

Ultrastructure and mineral distribution in the tergal cuticle of the terrestrial isopod Titanethes albus. Adaptations to a karst cave biotope

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.

Determination of Moult Stages of Porcellio Scaber (Isopoda) for Routine Use

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.

“Candidatus Bacilloplasma,” a Novel Lineage of Mollicutes Associated with the Hindgut Wall of the Terrestrial Isopod Porcellio scaber (Crustacea: Isopoda)

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.

Genetic diversity of bacteria associated with the hindgut of the terrestrial crustacean Porcellio scaber (Crustacea : Isopoda)

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.

Silicon: The key element in early stages of biocalcification.

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 earths 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.

Ultrastructural alterations of the hepatopancreas in Porcellio scaber under stress.

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.

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 crustaceanLigia 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.

Focused ion beam (FIB)/scanning electron microscopy (SEM) in tissue structural research

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.

The effect of Zn on the digestive gland epithelium ofPorceffio 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 μg 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 μg 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.