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Polyhydroxyalkanoates (PHA) are abundant microbial polyesters accumulated in the form of intracellular granules by numerous prokaryotes primarily as storage of carbon and energy. Apart from their storage function, the presence of PHA also enhances the robustness of the microbial cells against various stressors. In this work, we investigated the role of PHA in Cupriavidus necator, a model organism concerning PHA metabolism, for adaptation to osmotic pressure and copper ions. In long-term laboratory evolution experiments, the bacterial culture was cultivated in presence of elevated doses of sodium chloride or copper ions (incubations lasted 78 passages for Cu

Polyhydroxyalkanoates (PHA) are microbial polyesters that have recently come to the forefront of interest due to their biodegradability and production from renewable sources. A potential increase in competitiveness of PHA production process comes with a combination of the use of thermophilic bacteria with the mutual use of waste substrates. In this work, the thermophilic bacterium Tepidimonas&nbsp, taiwanensis LMG 22826 was identified as a promising PHA producer. The ability to produce PHA in T.&nbsp, taiwanensis was studied both on genotype and phenotype levels. The gene encoding the Class I PHA synthase, a crucial enzyme in PHA synthesis, was detected both by genome database search and by PCR. The microbial culture of T. taiwanensis was capable of efficient utilization of glucose and fructose. When cultivated on glucose as the only carbon source at 50°C, the PHA titers reached up to 3.55 g/L, and PHA content in cell dry mass was 65%. The preference of fructose and glucose opens the possibility to employ T. taiwanensis for PHA production on various food wastes rich in these abundant sugars. In this work, PHA production on grape pomace extracts was successfully tested.

In sample preparation of biological samples for electron microscopy, many types of embedding media are widely used. Unfortunately, none of them is perfectly resistant to beam induced damage. The article is focused on mass loss measuring of pure epoxy resin EMbed 812 that replaced Epon – the most widely used embedding resin for biological electron microscopy, in a form of ultrathin sections with thicknesses ranging from 30 to 100 nm. The STEM imaging was performed in a quantitative way which allowed us to estimate the mass loss directly up to the total dose of 3000 e−/nm2. For data acquisition we used SEM equipped with a commercial STEM detector working at a relatively low acceleration voltage of 30 kV. In this study we estimated the influence of various factors which can affect the endurance of the epoxy resin EMbed 812 ultrathin sections under an electron beam, such as the sample aging, differences between storing the samples in forms of ultrathin sections and whole blocks, ultrathin sections thicknesses, temperature of the sample, probe current, and one or two-sided carbon coating of ultrathin sections. The aim of this work is to investigate beam induced mass loss at electron energies of SEM and find out how to reduce the mass loss.

Aneurinibacillus sp. H1 is a promising, moderately thermophilic, novel Gram-positive bacterium capable of the biosynthesis of polyhydroxyalkanoates (PHA) with tunable monomer composition. In particular, the strain is able to synthesize copolymers of 3-hydroxybutyrate (3HB), 4-hydroxybutyrate (4HB) and 3-hydroxyvalerate (3HV) with remarkably high 4HB and 3HV fractions. In this study we performed an in-depth material analysis of PHA polymers produced by Aneurinibacillus sp. H1 in order to describe how the monomer composition affects fundamental structural and physicochemical parameters of the materials in the form of solvent-casted films. Results of infrared spectroscopy, X-ray diffractometry and thermal analysis clearly show that controlling the monomer composition enables optimization of PHA crystallinity both qualitatively (the type of the crystalline lattice) and quantitatively (the overall degree of crystallinity). Furthermore, resistance of the films against thermal and/or enzymatic degradation can also be manipulated by the monomer composition. Results of this study hence confirm Aneurinibacillus sp. H1 as an auspicious candidate for thermophilic production of PHA polymers with material properties that can be tuned together with their chemical composition by the corresponding adjustment of the cultivation process.

Polyhydroxyalkanoates (PHA) are storage polymers accumulated by numerous prokaryotes in form of intracellular granules. Native PHA granules are formed by amorphous polymer which reveals considerably higher elasticity and flexibility as compared to crystalline pure PHA polymers. The fact that bacteria store PHA in amorphous state has great biological consequences. It is not clear which mechanisms protect amorphous polymer in native granules from transition into thermodynamically favorable crystalline state. Here, we demonstrate that exposition of bacterial cells to particular stressors induces granules aggregation, which is the first but not sufficient condition for PHA crystallization. Crystallization of the polymer occurs only when the stressed bacterial cells are subsequently dried. The fact that both granules aggregation and cell drying must occur to induce crystallization of PHA indicates that both previously suggested hypotheses about mechanisms of stabilization of amorphous state of native PHA are valid and, in fact, both effects participate synergistically. It seems that the amorphous state of the polymer is stabilized kinetically by the low rate of crystallization in limited volume in small PHA granules and, moreover, water present in PHA granules seems to function as plasticizer protecting the polymer from crystallization, as confirmed experimentally for the first time by the present work.

In this study we present an innovative method for the preparation of fully hydrated samples of microbial biofilms of cultures Staphylococcus epidermidis, Candida parapsilosis and Candida albicans. Cryo-scanning electron microscopy (cryo-SEM) and high-pressure freezing (HPF) rank among cutting edge techniques in the electron microscopy of hydrated samples such as biofilms. However, the combination of these techniques is not always easily applicable. Therefore, we present a method of combining high-pressure freezing using EM PACT2 (Leica Microsystems), which fixes hydrated samples on small sapphire discs, with a high resolution SEM equipped with the widely used cryo-preparation system ALTO 2500 (Gatan). Using a holder developed in house, a freeze-fracturing technique was applied to image and investigate microbial cultures cultivated on the sapphire discs. In our experiments, we focused on the ultrastructure of the extracellular matrix produced during cultivation and the relationships among microbial cells in the biofilm. The main goal of our investigations was the detailed visualization of areas of the biofilm where the microbial cells adhere to the substrate/surface. We show the feasibility of this technique, which is clearly demonstrated in experiments with various freeze-etching times.

Extremophilic microorganisms are considered being very promising candidates for biotechnological production of various products including polyhydroxyalkanoates (PHA). The aim of this work was to evaluate the PHA production potential of a novel PHA-producing thermophilic Gram-positive isolate Aneurinibacillus sp. H1. This organism was capable of efficient conversion of glycerol into poly(3-hydroxybutyrate) (P3HB), the homopolyester of 3-hydroxybutyrate (3HB). In flasks experiment, under optimal cultivation temperature of 45 &deg, C, the P3HB content in biomass and P3HB titers reached 55.31% of cell dry mass and 2.03 g/L, respectively. Further, the isolate was capable of biosynthesis of PHA copolymers and terpolymers containing high molar fractions of 3-hydroxyvalerate (3HV) and 4-hydroxybutyrate (4HB). Especially 4HB contents in PHA were very high (up to 91 mol %) when 1,4-butanediol was used as a substrate. Based on these results, it can be stated that Aneurinibacillus sp. H1 is a very promising candidate for production of PHA with tailored material properties.

The biofilm-forming microbial species Candida parapsilosis and Staphylococcus epidermidis have been recently linked to serious infections associated with implanted medical devices. We studied microbial biofilms by high resolution scanning electron microscopy (SEM), which allowed us to visualize the biofilm structure, including the distribution of cells inside the extracellular matrix and the areas of surface adhesion. We compared classical SEM (chemically fixed samples) with cryogenic SEM, which employs physical sample preparation based on plunging the sample into various liquid cryogens, as well as high-pressure freezing (HPF). For imaging the biofilm interior, we applied the freeze-fracture technique. In this study, we show that the different means of sample preparation have a fundamental influence on the observed biofilm structure. We complemented the SEM observations with Raman spectroscopic analysis, which allowed us to assess the time-dependent chemical composition changes of the biofilm in vivo. We identified the individual spectral peaks of the biomolecules present in the biofilm and we employed principal component analysis (PCA) to follow the temporal development of the chemical composition.

Polyhydroxyalkanoates (PHA) are microbial polyesters which accumulate as intracellular granules in numerous prokaryotes and mainly serve as storage materials; beyond this primary function, PHA also enhance the robustness of bacteria against various stress factors. We have observed that the presence of PHA in bacterial cells substantially enhances their ability to maintain cell integrity when suddenly exposed to osmotic imbalances. In the case of the non-halophilic bacterium Cupriavidus necator, the presence of PHA decreased plasmolysis-induced cytoplasmic membrane damage during osmotic up-shock, which subsequently enabled the cells to withstand subsequent osmotic downshock. In contrast, sudden induction of osmotic up- and subsequent down-shock resulted in massive hypotonic lysis of non-PHA containing cells as determined by Transmission Electron Microscopy and Thermogravimetrical Analysis. Furthermore, a protective effect of PHA against hypotonic lysis was also observed in the case of the halophilic bacterium Halomonas halophila; here, challenged PHA-rich cells were capable of retaining cell integrity more effectively than their PHA-poor counterparts. Hence, it appears that the fact that PHA granules, as an added value to their primary storage function, protect halophiles from the harmful effect of osmotic down-shock might explain why PHA accumulation is such a common feature among halophilic prokaryotes. The results of this study, apart from their fundamental importance, are also of practical biotechnological significance: because PHA-rich bacterial cells are resistant to osmotic imbalances, they could be utilized in in-situ bioremediation technologies or during enrichment of mixed microbial consortia in PHA producers under conditions of fluctuating salinity.

The aim of this work was to study the potential of selected Halomonas species for conversion of waste frying oil into polyhydroxyalkanoates (PHA). In total nine Halomonas strains were experimentally screened for their capability of PHA production. Among them, Halomonas neptunia and Halomonas hydrothermalis were identified as potent PHA producers. Initial concentration of NaCl was identified as parameter influencing PHA yields as well as molecular weight of the polymer. In addition, H. hydrothermalis was capable of biosynthesis of a copolymer of 3-hydroxybutyrate and 3-hydroxyvalerate P(3HB-co-3HV). When valerate was utilized as a precursor, the 3HV fraction in the copolymer reached high values of 50.15 mol.%. PHA production on lipid substrates by Halomonas has not been reported so far. Bearing in mind all the positive aspects of employing extremophiles in industrial biotechnology, H. hydrothermalis seems to be a very interesting halophilic strain for production of PHA using lipid substrates.

The ultrastructure and morphology of beluga spermatozoa (Huso huso) (Acipenseridae, Chondrostei) were studied by scanning and transmission electron microscopy. Spermatozoa of 51.27 ± 4.71 μm total length (mean ± SD) were typical of sturgeon and paddlefish spermatozoa. Each consisted of an elongated nucleus (length: 5.84 ± 0.46 μm) with distinct acrosome, a cylindrical midpiece (length: 2.10 ± 0.42 μm), and a single flagellum (length: 42.21 ± 3.82 μm). The acrosome was umbrella shaped (length: 1.12 ± 0.14 μm, width: 0.87 ± 0.10 μm) with seven to nine posterolateral projections (length: 0.49 ± 0.09 μm). Three endonuclear canals, spirally arranged, traversed the nucleus length from the acrosome to the implantation fossa. The flagellum comprised an axoneme with the typical 9+2 organization of microtubules. Two flat fins were present along the flagellum parallel to the plane of the central doublet of microtubules. These fins arose at different positions, 0.57 ± 0.30 and 4.06 ± 1.32 μm from the midpiece and terminated 4.18 ± 1.09 and 4.92 ± 1.34 μm from the distal end of the flagellum. Principal component analysis revealed spermatozoan ultrastructure and morphology were similar among sturgeon species, and, although assigned to genus Huso, beluga may be closely related to the genus Acipenser.

Numerous prokaryotes accumulate polyhydroxybutyrate (PHB) intracellularly as a storage material. It has also been proposed that PHB accumulation improves bacterial stress resistance. Cupriavidus necator and its PHB non-accumulating mutant were employed to investigate the protective role of PHB under hypertonic conditions. The presence of PHB granules enhanced survival of the bacteria after exposure to hypertonic conditions. Surprisingly, when coping with such conditions, the bacteria did not utilize PHB to harvest carbon or energy, suggesting that, in the osmotic upshock of C. necator , the protective mechanism of PHB granules is not associated with their hydrolysis. The presence of PHB granules influenced the overall properties of the cells, since challenged PHB-free cells underwent massive plasmolysis accompanied by damage to the cell membrane and the leakage of cytoplasm content, while no such effects were observed in PHB containing bacteria. Moreover, PHB granules demonstrated “liquid-like” properties indicating that they can partially repair and stabilize cell membranes by plugging small gaps formed during plasmolysis. In addition, the level of dehydration and changes in intracellular pH in osmotically challenged cells were less pronounced for PHB-containing cultures, demonstrating the important role of PHB for bacterial survival under hyperosmotic conditions.

Stable Pd nanocubes (PdNC) with the average size ∼15 nm were prepared by the controlled reduction of sodium tetrachloropalladate with ascorbic acid in water, in the presence of polyvinylpyrrolidone and potassium bromide. Morphology of the particles was characterized by transmission electron microscopy (TEM) and their stability in the colloidal solution was verified by dynamic light scattering (DLS). It has been demonstrated that the Pd nanocubes can be distinguished from commercial Au nanospheres in a standard TEM microscope by means of automated image analysis. In the next step, the PdNC were successfully conjugated to immunoglobulin proteins and used for the detection of a specific protein (nucleophosmin) on ultrathin sections of HeLa cells. Our experiments showed that PdNC can be used for multiple immunolabeling in combination with commercial Au nanospheres.

We describe the detection of sialylated N-linked glycans in partially fed Ixodes ricinus tick females using matrix-assisted laser desorption/ionization time-of-flight/time-of-flight mass spectrometry. Sialylated glycans were detected in salivary glands as well as in tick guts and we propose the host origin of these structures. In addition, we mapped the transport of sialylated structures from the blood meal through the gut to the salivary glands using electron microscopy. Specific localization of sialylated glycans to basement membranes of salivary glands was observed. Finally, the influence of the sample preparation methods for electron microscopy on ultrastructure and immunogold labeling was evaluated.

The progamic phase of male gametophyte development involves activation of synthetic and catabolic processes required for the rapid growth of the pollen tube. It is well-established that both transcription and translation play an important role in global and specific gene expression patterns during pollen maturation. On the contrary, germination of many pollen species has been shown to be largely independent of transcription but vitally dependent on translation of stored mRNAs. Here, we report the first structural and proteomic data about large ribonucleoprotein particles (EPPs) in tobacco male gametophyte. These complexes are formed in immature pollen where they contain translationally silent mRNAs. Although massively activated at the early progamic phase, they also serve as a long-term storage of mRNA transported along with the translational machinery to the tip region. Moreover, EPPs were shown to contain ribosomal subunits, rRNAs and a set of mRNAs. Presented results extend our view of EPP complexes from mere RNA storage and transport compartment in particular stages of pollen development to the complex and well-organized machinery devoted to mRNA storage, transport and subsequent controlled activation resulting in protein synthesis, processing and precise localization. Such an organization is extremely useful in fast tip-growing pollen tube. There, massive and orchestrated protein synthesis, processing, and transport must take place in accurately localized regions. Moreover, presented complex role of EPPs in tobacco cytoplasmic mRNA and protein metabolism makes them likely to be active in another plant species too. Expression of vast majority of the closest orthologues of EPP proteins also in Arabidopsis male gametophyte further extends this concept from tobacco to Arabidopsis, the model species with advanced tricellular pollen.

Summary The present study was carried out with two sturgeon species, Siberian sturgeon (Acipenser baerii) and sterlet (A. ruthenus) to compare spermatological parameters to better understand inter-species differences. Significant differences between morphometric features were observed such as acrosome length, acrosome width, head length, midpiece width and flagellar length, while midpiece length did not reveal such differences. The sterlet has a shorter spermatozoon than the Siberian sturgeon. Ultrastructural parameters vary significantly in terms of length of the nucleus, diameter of the endonuclear canals (EC), size of posterolateral projections (PP) and diameter of flagellum. Mean values for density of spermatozoa in the semen, seminal plasma pH, osmolality (mOsmol kg−1), along with Ca2+, Na+, K+, Cl− ions concentrations (mm) were determined to be 0.61 ± 0.37 × 109, 8.16 ± 0.18, 77.20 ± 52.28, 0.24 ± 0.06, 31.39 ± 10.21, 3.51 ± 1.10, 14.00 ± 4.30 in A. baerii and 0.41 ± 0.32 × 109, 8.13 ± 0.19, 50.74 ± 6.27, 0.16 ± 0.11, 20.11 ± 3.78, 1.26 ± 0.54, 6.11 ± 0.60 in A. ruthenus, respectively. Significant differences were observed in Na+, K+ and Cl− concentrations in the seminal plasma as well as in sperm velocity. The percentage of motile spermatozoa did not show any significant difference between the two species. Comparing the results of this study with published literature data on sturgeon spermatozoa reveals that morphological and ultrastructural parameters of spermatozoa together with some parameters of the seminal fluid and spermatozoa velocity can be used in comparative spermatology to better understand inter-species differences. The observed biochemical and physiological differences should be also considered for the development of methods for controlled reproduction and for sperm cryopreservation techniques.

Structure and organisation of Photosystem I and Photosystem II isolated from red alga Cyanidium caldarium was determined by electron microscopy and single particle image analysis. The overall structure of Photosystem II was found to be similar to that known from cyanobacteria. The location of additional 20 kDa (PsbQ′) extrinsic protein that forms part of the oxygen evolving complex was suggested to be in the vicinity of cytochrome c-550 (PsbV) and the 12 kDa (PsbU) protein. Photosystem I was determined as a monomeric unit consisting of PsaA/B core complex with varying amounts of antenna subunits attached. The number of these subunits was seen to be dependent on the light conditions used during cell cultivation. The role of PsaH and PsaG proteins of Photosystem I in trimerisation and antennae complexes binding is discussed.

Background information. Available data concerning the sperm morphology of teleost fishes demonstrate wide variation. In the present study, the spermatozoa of Siberian sturgeon (Acipenser baerii Brandt, 1869), a chondrostean fish, was investigated. In contrast with teleost fish, chondrostean spermatozoa have a head with a distinct acrosome, whereas other structures, such as a midpiece and a single flagellum, are present in spermatozoa of most species. Results. The average length of the head including the acrosome and the midpiece was 7.01±0.83 μm. Ten posterolateral projections derived from the acrosome were present on a subacrosomal region, with mean lengths of 0.94±0.15 μm and widths of 0.93±0.11 μm. The nucleus consisted of electrodense homogeneous nuclear chromatin. Three intertwining endonuclear canals, bound by membranes, traversed the nucleus longitudinally from the acrosomal end to the basal nuclear fossa region. There were between three and six mitochondria, two types of centrioles (proximal and distal) in the midpiece and two vacuoles composed of lipid droplets. The flagellum (44.75±4.93 μm in length), originating from the centriolar apparatus, had a typical 9+2 eukaryotic flagellar organization. In addition, there was an extracellular cytoplasm canal between the cytoplasmic sheath and the flagellum. Conclusions. A principal components analysis explained the individual morphological variation fairly well. Of the total accumulated variance, 41.45% was accounted for by parameters related to the head and midpiece of the sperm and the length of the flagellum. Comparing the present study with previous studies of morphology of sturgeon spermatozoa, there were large inter- or intra-specific differences that could be valuable taxonomically.

We present a new method of multiple immunolabeling that is suitable for a broad spectrum of biomedical applications. The general concept is to label both sides of the ultrathin section with the thickness of 70-80 nm with different antibodies conjugated to gold nanoparticles and to distinguish the labeled side by advanced imaging methods with high resolution scanning electron microscopy, such as by correlating images acquired at different energies of primary electrons using different signals. From the Clinical Editor: The use of transmission electron microscopy has become an indispensible tool in the detection of cellular proteins. In this short but interesting article, the authors described their new method of labeling and the identification of four different proteins simultaneously, which represents another advance in imaging technique.

Cyanobacteria contain several genes coding for small one-helix proteins called SCPs or HLIPs with significant sequence similarity to chlorophyll a/b-binding proteins. To localize one of these proteins, ScpD, in the cells of the cyanobacterium Synechocystis sp. PCC 6803, we constructed several mutants in which ScpD was expressed as a His-tagged protein (ScpDHis). Using two-dimensional native-SDS electrophoresis of thylakoid membranes or isolated Photosystem II (PSII), we determined that after high-light treatment most of the ScpDHis protein in a cell is associated with PSII. The ScpDHis protein was present in both monomeric and dimeric PSII core complexes and also in the core subcomplex lacking CP43. However, the association with PSII was abolished in the mutant lacking the PSII subunit PsbH. In a PSII mutant lacking cytochrome b(559), which does not accumulate PSII, ScpDHis is associated with CP47. The interaction of ScpDHis with PsbH and CP47 was further confirmed by electron microscopy of PSII labeled with Ni-NTA Nanogold. Single particle image analysis identified the location of the labeled ScpDHis at the periphery of the PSII core complex in the vicinity of the PsbH and CP47. Because of the fact that ScpDHis did not form any large structures bound to PSII and because of its accumulation in PSII subcomplexes containing CP47 and PsbH we suggest that ScpD is involved in a process of PSII assembly/repair during the turnover of pigment-binding proteins, particularly CP47.

The tench Tinca tinca is an interesting fish from the viewpoint of polyploidy and related atypical reproduction aspects. Triploid tench were produced artificially. Studies of spermiation as well as of sperm motility and structure were performed on several triploid and diploid males simultaneously with individual experimental crosses with diploid females to define their reproductive capacities. The testes of triploids visually looked less developed in the most of cases with lower sperm production (0.05 cm3 sperm per male), GSI and weight of testes compared to diploids (0.58 cm3 sperm per male). Analysis of variance showed significant influence of ploidy level on the percentage of motile spermatozoa. Triploidy did not change percentage of live spermatozoa and velocity of spermatozoa at the first time of sperm movement. The study of sperm structure by scanning electron microscopy revealed that most sperm cells were of normal structure with some anomalies. Sperm heads of triploid and diploid males were mostly round-shaped, 1.86±0.2 and 1.6±0.18 μm in diameter. The midpiece of triploid spermatozoa was slightly narrower than that of diploid ones with typical cylindrical shape. Flow cytometry revealed sperm cells of triploids to be largely aneuploid (1.47 n) with high mosaic DNA, oscillating from haploid DNA content (1.0 n) to diploid DNA content (1.9 n). Experimental crosses between triploid males and diploid females revealed that these males were capable to stimulate effective development with relatively high level of fertilization and hatching rates from 0 to 70%. In conclusion, triploidization does not seem to guarantee sterility of tench.

In the apical glandular region of the adult Proteocephalus torulosus (Batsch, 1786), two types of eccrine gland cells are present. The first type of unicellular gland produces large electron-dense granules of various sizes. The second type contains small electron-dense granules. Most cells form glands with large granules; glands with small granules are infrequent. The secretion of both types of gland cells is concentrated in the apical parts of the cyton and in the ducts opening to the exterior. On the scolex of P. torulosus, there are regional structural differences of the microthrix border. The apical glandular region bears filamentous microtriches only. On the remaining frontal part, surrounding the glandular region, there are blade-like and filamentous microtriches. The lateral parts of the scolex and suckers bear blade-like microtriches. Possible functions of both types of gland cells and different parts of the scolex microthrix border are discussed. The unique structure of the frontal part of the scolex of P. torulosus and its differences from Proteocephalus macrocephalus, P. longicollis and P. percae correlate well with the putative basal phylogenetic position of P. torulosus among European species of Proteocephalus.

The reinvestigation of spermiogenesis in the proteocephalidean cestode Proteocephalus longicollis (Zeder, 1800) provides new details that were not described previously for this species. The 2 centrioles with roots are oriented at the first tangential to the long axis of the nucleus in a differentiation zone. They come to be arranged in the same plane only when a single intercentriolar body (ICB) appears between them. The ICB had been described previously as consisting of a number of dense elements (Swiderski, 1985) but, in the present study, appeared as a single, electron-dense plate. Also new for this species is the observation of 2 arching membranes that appear at the base of the differentiation zone only when the 2 flagella rotate toward the median cytoplasmic protrusion. Moreover, the formation of a crested body is described for the first time in P. longicollis. The present data support the suggestion that the process of spermiogenesis in proteocephalidean tapeworms corresponds in most features to that observed in some members of the unrelated tetraphyllidean clades, the Onchobothriidae and Phyllobothriidae. This may represent more evidence regarding the close relationship between tetraphyllidean and proteocephalidean cestodes, but existing information is fragmentary and controversial in some characters.

In the adult fish trematode Crepidostomum metoecus (Braun, 1900), four types of sensory receptors were observed inside the forebody tegument and one type beneath the tegument basal lamina. Two types of sensory receptors extend through the thickness of tegument and have a free cilium inside a pit (types I and II). Two types (III and IV) are nonciliate and entirely intra-tegumental in location. Type IV receptor with large horizontal and thin vertical rootlets was described earlier in aspidogastreans only. Below the basal lamina, nerve endings in close association with muscle fibres, comparable with those in the Aspidogastrea, were detected.

The neodermis of the whole early rostellum of Silurotaenia siluri (Batsch, 1786) bears filamentous microtriches. At the base, there are five to six irregular rows of hooks and spine-like microtriches. The rostellar hooks of S. siluri originate through the enlargement of microtriches. Electron-dense hook substance is deposited along the edges of the microthrix to form the hook blade and basal plate. The blade does not become hollow as in the cyclophyllidean cestodes. The basal plate of the hook, corresponding to the handle and the guard of Cyclophyllidea, is formed by the deposition of hook substance around the base of the microthrix. Within the centre of the base, only a narrow cleft-like core is occupied by distal cytoplasm. The hook bases are more deeply inserted into the distal cytoplasm, as the bases of the spine-like microtriches are localized on the rostellum below the rows of hooks. No hemidesmosomes, fixing the hook base to the basal lamina, are present. Eccrine gland cells and uniciliate and nonciliate sensory receptors of the primitive rostellum are described.

The forebody and foregut of Crepidostomum metoecus Braun, 1900 are invested with a tegument bearing regularly arranged surface tubercles comparable with the aspidogastrean surface structures. The tegument of the ventrolateral lobes and of the prepharynx is penetrated by ducts of eccrine gland cells. The frontal and prepharyngeal gland cells, localised in the parenchyma, discharge electron-dense granules. Their ducts are lined by peripheral microtubules and fixed to the tegument plasmalemma by a septate junction. The functional roles of these glands are discussed.

Le stichosome (partie posterieure glandulaire de l'œsophage) de Capillaria pterophylli Heinze, 1933 est forme de grandes cellules glandulaires (stichocytes) et d'un epithelium revetu de cuticule entourant la lumiere œsophagienne. Ces deux structures sont enfermees dans un reseau de cellules musculaires. Le cytoplasme du stichocyte contient des petites citernes de reticulum endoplasmique rugueux, des complexes de Golgi, un seul type de granules de secretion denses aux electrons, des mitochondries et un systeme ramifie de canaux collecteurs intracellulaires qui ne sont pas entoures de faisceaux de filaments.

When developing new nanoparticles for bio-applications, it is important to fully characterize the nanoparticle's behavior in biological systems. The most common techniques employed for mapping nanoparticles inside cells include transmission electron microscopy (TEM) and scanning transmission electron microscopy (STEM). These techniques entail passing an electron beam through a thin specimen. STEM or TEM imaging is often used for the detection of nanoparticles inside cellular organelles. However, lengthy sample preparation is required (i.e., fixation, dehydration, drying, resin embedding, and cutting). In the present work, a new matrix (FTO glass) for biological samples was used and characterized by field emission scanning electron microscopy (FE-SEM) to generate images comparable to those obtained by TEM. Using FE-SEM, nanoparticle images were acquired inside endo/lysosomes without disruption of the cellular shape. Furthermore, the initial steps of nanoparticle incorporation into the cells were captured. In addition, the conductive FTO glass endowed the sample with high stability under the required accelerating voltage. Owing to these features of the sample, further analyses could be performed (material contrast and energy-dispersive X-ray spectroscopy (EDS)), which confirmed the presence of nanoparticles inside the cells. The results showed that FE-SEM can enable detailed characterization of nanoparticles in endosomes without the need for contrast staining or metal coating of the sample. Images showing the intracellular distribution of nanoparticles together with cellular morphology can give important information on the biocompatibility and demonstrate the potential of nanoparticle utilization in medicine.

The fine structure of the mature spermatozoon of the pseudophyllidean tapeworm Eubothrium crassum, a parasite of salmonid fishes, has been studied by transmission electron microscopy for the first time. The mature spermatozoon of E. crassum is filiform and tapered at both extremities. It contains two axonemes of unequal length showing the 9 + "1" pattern of Trepaxonemata. The anterior extremity exhibits a crested body 50-100 nm thick. It spirals around the outside of the anterior region of the spermatozoon. The nucleus is electron-dense, exhibiting a fibrous appearance in its middle (the broadest) region. The cortical microtubules are of two types and are situated parallel to the spermatozoon axis. The cytoplasm is slightly electron-dense and contains numerous electron-dense granules in region II of the spermatozoon. A ring of electron-dense, centred microtubules surrounds the axoneme, together with the underlying ring of moderately electron-dense, subjacent submicrotubular material in region V. The anterior and posterior extremities of the spermatozoon lack cortical microtubules and contain a single axoneme. Our results reveal several peculiarities, in which the spermatozoon of E. crassum differs from those of other pseudophyllidean cestodes.

In the scolex-neck region of the adult Proteocephalus macrocephalus two types of eccrine gland cells are present. The first type of gland cells, localized in the frontal part of the scolex only, contains large, more or less round electron lucid granules. The second type of unicellular glands produces large electron dense granules. These electron dense granular gland cells are localized primarily in the neck region, only few are present in the scolex apex. The secretion of both types of gland cells is concentrated in the ducts opening to the exterior. The ducts are fixed to the plasmalemma of the tegument by septate junctions. The function of both types of gland cells is discussed.

The secondary osmoregulatory canals in the scolex and neck region of Silurotaenia siluri, a parasite of the catfish Silurus glanis (L.), terminate below the tegument basal plasma membrane. The basal plasma membrane of the osmoregulatory canal syncytium is in tight contact with the tegument basal plasma membrane.

Many bacteria are capable of accumulating intracellular granules of polyhydroxyalkanoates (PHA). In this work, we developed confocal microscopy analysis of bacterial cells to study changes in the diameters of cells as well as PHA granules during growth and PHA accumulation in the bacterium Cupriavidus necator H16 (formerly Ralstonia eutropha). The cell envelope was stained by DiD(®) fluorescent probe and PHA granules by Nile Red. Signals from both probes were separated based on their spectral and fluorescence life-time properties. During growth and PHA accumulation, bacterial cells increased their length but the width of the cells remained constant. The volume fraction of PHA granules in cells increased during PHA accumulation, nevertheless, its value did not exceed 40 vol. % regardless of the PHA weight content. It seems that bacterial cultures lengthen the cells in order to control the PHA volume portion. However, since similar changes in cell length were also observed in a PHA non-accumulating mutant, it seems that there is no direct control mechanism, which regulates the prolongation of the cells with respect to PHA granules volume. It is more likely that PHA biosynthesis and the length of cells are influenced by the same external stimuli such as nutrient limitation.

The possibilities of using a field emission scanning electron microscope (FESEM) equipped with an Autrata improved yttrium aluminum garnet (YAG) detector of back-scattered electrons (BSE) for a determination of metal nanoparticles sizes were examined in this study. The diameters of Au and Pd nanoparticles with different sizes were resolved based on digitally recorded FESEM micrographs and were compared with diameters obtained with a transmission electron microscope (TEM). In the case of Au nanoparticles, the data obtained from the FESEM exhibited smaller values in comparison with data from the TEM. The Pd nanoparticles diameters measured in the FESEM were comparable with those determined by means of the TEM, however. The influence of various parameters like the sizes of nanoparticles, their composition, use of accelerating voltage and other working conditions in the FESEM on the measurement results are discussed.

Seven microsporidian species infecting caddis fly larvae, corresponding to conventional genera Episeptum, Pyrotheca and Cougourdella were studied using light and electron microscopy. Parts of their small subunit, ITS and large subunit ribosomal RNA genes were sequenced and compared with sequences of rDNA obtained from syntype slides of Cougourdella polycentropi Weiser 1965 and Pyrotheca sp. from Hydropsyche pellucidula. All studied caddis fly microsporidia form a closely related group. Their developmental stages in trichopteran hosts are restricted to fat body cells and oenocytes and have isolated nuclei. In late merogony, uninucleate meronts and binucleate plasmodia are formed. In sporogony a sporogonial plasmodium with four nuclei gives rise by rosette-like budding to four sporoblasts within a non-persistent sporophorous vesicle. Sporoblasts mature into pyriform to lageniform spores. The shape and size of spores, the number of polar filament coils, the structure of the polaroplast and of the exospore, together with morphometric characters present a set of markers unique for respective species. Four new species are established. The new genus Paraepiseptum is proposed to replace the tetrasporoblastic Pyrotheca and Cougourdella species from caddis flies. The genus Episeptum is redefined. Field and laboratory examinations as well as the phylogenetic position within the aquatic clade of microsporidia suggest that the life cycle of trichopteran microsporidia probably involves an alternate (copepod?) host and (or) transovarial transmission.

Mitochondria are archetypal organelles of endosymbiotic origin in eukaryotic cells. Some unicellular eukaryotes (protists) were considered to be primarily amitochondrial organisms that diverged from the eukaryotic lineage before the acquisition of the premitochondrial endosymbiont, but their amitochondrial status was recently challenged by the discovery of mitochondria-like double membrane-bound organelles called mitosomes. Here, we report that proteins targeted into mitosomes of Giardia intestinalis have targeting signals necessary and sufficient to be recognized by the mitosomal protein import machinery. Expression of these mitosomal proteins in Trichomonas vaginalis results in targeting to hydrogenosomes, a hydrogen-producing form of mitochondria. We identify, in Giardia and Trichomonas , proteins related to the component of the translocase in the inner membrane from mitochondria and the processing peptidase. A shared mode of protein targeting supports the hypothesis that mitosomes, hydrogenosomes, and mitochondria represent different forms of the same fundamental organelle having evolved under distinct selection pressures.