Spermiogenesis produces the spermatozoa with 9 + '1' and 9 + 0 axonemal pattern in progenetic cestode Diplocotyle olrikii Krabbe, 1874 (Spathebothriidea: Acrobothriidae).

Spermiogenesis in the progenetic spathebothriidean cestode Diplocotyle olrikii has been examined using transmission electron microscopy (TEM) for the first time. Along with the typical features of spermatozoon cytodifferentiation (e.g., the electron-dense material in the apical region of the differentiation zone in the early stage of spermiogenesis, the intercentriolar body which is composed of three electron-dense plates and two electron-lucent zones, the orthogonal development of the two flagella, a flagellar rotation, proximo-distal fusion, the presence of two pairs of electron-dense attachment zones), new for the Eucestoda is detection of the formation of two types of free flagella during spermiogenesis in progenetic D. olrikii, exhibiting either standard 9 + '1' trepaxonematan pattern, or atypical 9 + 0 structure. Various combinations of these two types of flagella resulted in the production of three types of male gametes during spermiogenesis in this spathebothriidean cestode. The first type is represented with the two axonemes of the 9 + '1' structure; the second type exhibits two different axonemes, i.e., one with 9 + '1' and the other of 9 + 0 pattern; and the third type has two axonemes with atypical 9 + 0 structure. The occurrence of three sperm types in progenetic D. olrikii is associated with typical spermiogenesis and has never been described previously in the Platyhelminthes. We suppose that heteromorphism of male gametes in D. olrikii might be linked to progenesis, i.e., the programmed sexual maturation detected during the larval/developmental stage of an organism.

Heteromorphism of sperm axonemes in a parasitic flatworm, progenetic Diplocotyle olrikii Krabbe, 1874 (Cestoda, Spathebothriidea).

The spermatozoon ultrastructure of the progenetic cestode Diplocotyle olrikii (Spathebothriidea) has been examined using transmission electron microscopy and cytochemical staining with periodic acid-thiosemicarbazide-silver proteinate (PA-TSC-SP) for glycogen. The spermatozoon is a filiform cell, tapered at both extremities. Its moderately electron-dense cytoplasm possesses two parallel axonemes of unequal lengths. New for the Cestoda is a finding of three types of the mature spermatozoa with respect to different axonemal structure. The first type has both axonemes with standard 9 + '1' trepaxonematan pattern. The second type is represented by a spermatozoon having one axoneme with 9 + '1' structure and the second one with 9 + 0 pattern. The third type includes the two axonemes with 9 + 0 pattern. Microtubule doublets of the 9 + 0 axonemes contain either inner dynein arms or no dynein arms. In addition to the two axonemes, all three types of the mature sperm cells contain parallel nucleus, parallel cortical microtubules, four electron-dense plaques/attachment zones, and glycogen. The anterior extremity of the gamete exhibits a centriole surrounded by a semiarc of up to five electron-dense tubular structures. The distal end of the first type spermatozoa exhibits two morphological variants, represented either by (i) nucleus or (ii) remnants of the disorganized axoneme. Distal extremity of the spermatozoa of the second and third types contains doublets and singlets of disorganized axoneme. The ultrastructural characters of the spermatozoon of progenetic D. olrikii support the basal position of the Spathebothriidea within the Eucestoda.

What keeps polyhydroxyalkanoates in bacterial cells amorphous? A derivation from stress exposure experiments.

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.

First ultrastructural and cytochemical data on the spermatozoon and its differentiation in progenetic and adult Archigetes sieboldi Leuckart, 1878 (Cestoda, Caryophyllidea, Caryophyllaeidae).

Spermiogenesis in progenetic and adult stages of Archigetes sieboldi Leuckart, 1878, a tapeworm parasitic in oligochaetes and fish respectively, has been examined using transmission electron microscopy and cytochemical staining for glycogen. General pattern of spermiogenesis is essentially like that of other caryophyllideans, i.e., apical dense material in the zone of differentiation in the early stages of spermiogenesis, rotation of free flagellum and a flagellar bud, and proximo-distal fusion. Interestingly, rotation of a free flagellum and flagellar bud to the median cytoplasmic process (MCP) has been observed unconventionally at > 90° only in progenetic stages. Typical striated roots associated with the centrioles occur rarely in A. sieboldi, and only in form of faint structures in advanced stages of spermiogenesis. In contrast to most caryophyllideans studied to date, penetration of the nucleus into the spermatid body has started before the fusion of the free flagellum with the MCP. This feature has been reported rarely but exclusively in the family Caryophyllaeidae. The unipartite mature spermatozoon of A. sieboldi is composed of one axoneme of the 9 + '1' trepaxonematan pattern with its centriole, parallel nucleus, and parallel cortical microtubules which are situated in a moderately electron-dense cytoplasm with glycogen particles. An unusual arrangement of cortical microtubules in the two parallel rows in region I of the spermatozoon is described here for the first time in the Caryophyllidea. Ultrastructural data on spermiogenesis and the spermatozoon in A. sieboldi from tubuficids and carp are compared and discussed with those in other caryophyllideans and/or Neodermata.

Ultrastructural, cytochemistry and electron tomography analysis of Caryophyllaeides fennica (Schneider, 1902) (Cestoda: Lytocestidae) reveals novel spermatology characteristics in the Eucestoda.

Spermatozoon formation in Caryophyllaeides fennica (Schneider, 1902) is characterised by the following: (1) apical electron-dense material in the zone of differentiation, (2) typical striated roots situated unconventionally in opposite directions in early stages of spermiogenesis, (3) intercentriolar body composed of three electron-dense and two electron-lucent plates, (4) free flagellum and a flagellar bud that correspond to a greatly reduced flagellum and (5) rotation of free flagellum and a flagellar bud to the median cytoplasmic process at 90°. The development of two flagella of significantly unequal length clearly supports a derived form of spermiogenesis in the Caryophyllidea. New for cestodes is a finding of two additional striated roots situated opposite each other, in conjunction with both the flagellar bud and free flagellum. Mutual position of additional striated roots and typical striated roots is parallel in early stages and perpendicular in advanced stages of spermiogenesis. A complete proximodistal fusion gives rise to a mature spermatozoon consisting of one axoneme, parallel cortical microtubules, a nucleus and a moderately electron-dense cytoplasm with glycogen particles, detected by a technique of Thiéry (J Microsc 6:987-1018, 1967), in the principal regions (II, III, IV). Electron tomography analysis of the free flagellum and one axoneme of a mature spermatozoon of C. fennica provides clear evidence, for the first time, that two tubular structures are present in the central axonemal electron-dense core. Phylogenetically important aspects of spermiogenesis of the Caryophyllidea with one axoneme, and other cestodes with one or two axonemes, are briefly reviewed and discussed.

The effect of rework content addition on the microstructure and viscoelastic properties of processed cheese.

The aim of this work was to add various amounts of rework (0.0 to 20.0% wt/wt) to processed cheeses with a dry matter content of 36% (wt/wt) and fat with a dry matter content of 45% (wt/wt). The effect of the rework addition on the viscoelastic properties and microstructure of the processed cheeses was observed. The addition of rework (in this case, to processed cheese with a spreadable consistency) in the amounts of 2.5, 5.0, and 10.0% (wt/wt) increased the firmness of the processed cheese. With the further addition of rework, the consistency of the processed cheeses no longer differed significantly. The conclusions obtained by the measurement of viscoelastic properties were supported by cryo-scanning electron microscopy, where fat droplets in samples with added rework of over 10.0% (wt/wt) were smaller than fat droplets in processed cheeses with lower additions of rework.

Monitoring Candida parapsilosis and Staphylococcus epidermidis Biofilms by a Combination of Scanning Electron Microscopy and Raman Spectroscopy.

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.

Microstructure and textural and viscoelastic properties of model processed cheese with different dry matter and fat in dry matter content.

The aim of this work was to examine the effect of a different dry matter (DM) contents (35 and 45% wt/wt) and fat in DM contents (40 and 50% wt/wt) on the textural and viscoelastic properties and microstructure of model processed cheeses made from real ingredients regularly used in the dairy industry. A constant DM content and constant fat in DM content were kept throughout the whole study. Apart from the basic chemical parameters, textural and viscoelastic properties of the model samples were measured and scanning electron microscopy was carried out. With increasing DM content, the rigidity of the products increased and the size of the fat globules in the model samples of the processed cheeses decreased. With increasing fat in DM content, the rigidity of the processed cheeses decreased and the size of the fat globules increased.

Occurrence, pathology, and ultrastructure of iridovirus and cytoplasmic polyhedrosis viruses in daphnids from the Czech Republic.

Iridescent (IVs, family Iridoviridae, genus Iridovirus) and cytoplasmic polyhedrosis viruses (CPVs; family Reoviridae, genus Cypovirus) are well known in insects, with thirteen IV species recognized from various orders, and sixteen CPV species known from lepidopterans. In 1975, an IV and CPV were reported in the daphnid, Simocehpalus expinosus, in Florida, but other reported daphnid virus infections seem to be rare. Here we report infected daphnids from woodland and carp ponds in the Czech Republic, Daphnia curvirostris with an IV, and D. pulex and D. ambigua, with CPVs. This suggests these viruses are more common in daphnids, the rarity of reports due to few surveys.

Globulispora mitoportans n. g., n. sp., (Opisthosporidia: Microsporidia) a microsporidian parasite of daphnids with unusual spore organization and prominent mitosome-like vesicles.

The microsporidian parasite Globulispora mitoportans, n. g., n. sp., infects the intestinal epithelium of two species of daphnids (Crustacea: Cladocera). Mature spores are thin-walled and possess a novel type of polaroplast with a conspicuous part consisting of globules that occupies a large part of the spore volume. Both developmental stages and the spores possess large, electron-lucent vesicles enveloped by a double membrane and filled with an internal web of filamentous material, corresponding structurally to microsporidian mitosomes. The SSU rRNA phylogeny places Globulispora into a specific "Enterocytospora-like" clade, part of a large "non-enterocytozoonidae" clade, grouping a heterogenous assemblage of microsporidia infecting almost exclusively insects and crustacea.

Novel method of simultaneous multiple immunogold localization on resin sections in high resolution scanning electron microscopy.

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.

Nippotaenia mogurndae Yamaguti et Myiata, 1940 (Cestoda, Nippotaeniidea): first data on spermiogenesis and sperm ultrastructure.

Spermiogenesis and the spermatozoon ultrastructure of the cestode Nippotaenia mogurndae Yamaguti et Myiata, 1940 (Nippotaeniidea), a parasite of Perccottus glenii Dubowski, 1877 (Perciformes: Odontobutidae), have been investigated by means of transmission electron microscopy, cytochemical staining with periodic acid-thiosemicarbazide-silver proteinate (PA-TSC-SP) for glycogen, and electron tomography. The process of spermatozoon formation is characterised by the presence of (1) two centrioles without typical striated rootlets, (2) a single intercentriolar body, (3) a flagellar rotation (free flagellum plus flagellar bud), and (4) a complete proximodistal fusion. The mature spermatozoon of N. mogurndae contains a single helicoidal crested body, one axoneme of the 9 + "1" trepaxonematan structure, parallel cortical microtubules arranged in a ring in the anterior region of the cell, and a spiraled nucleus encircling the axoneme. Intracellular components are situated in a moderately electron-dense cytoplasm, containing glycogen in the principal regions (II, III, IV) of the spermatozoon. Application of electron tomography has revealed a helicoidal nature of the central electron-dense core in the central cylinder of the axoneme in parasitic cestodes for the first time. The patterns of spermiogenesis and spermatozoon ultrastructure resemble most closely those in mesocestoidids and may reflect the relationships between Nippotaeniidea and Mesocestoididae.

An advanced fast method for the evaluation of multiple immunolabelling using gold nanoparticles based on low-energy STEM.

We present a powerful method for the simultaneous detection of Au nanoparticles located on both sides of ultrathin sections. The method employs a high-resolution scanning electron microscope (HRSEM) operating in scanning transmission electron microscopy (STEM) mode in combination with the detection of backscattered electrons (BSE). The images are recorded simultaneously during STEM and BSE imaging at the precisely selected accelerating voltage. Under proper imaging conditions, the positions of Au nanoparticles on the top or bottom sides can be clearly differentiated, hence showing this method to be suitable for multiple immunolabelling using Au nanoparticles (NPs) as markers. The difference between the upper and lower Au NPs is so large that it is possible to apply common software tools (such as ImageJ) to enable their automatic differentiation. The effects of the section thickness, detector settings and accelerating voltage on the resulting image are shown. Our experimental results correspond to the results modelled in silico by Monte Carlo (MC) simulations.

A Comparison of the Effects of Continuous Illumination and Day/Night Regimes on PHB Accumulation in Synechocystis Cells.

Poly(3-hydroxybutyrate) (PHB) is a biobased and biodegradable polymer with properties comparable to polypropylene and therefore has the potential to replace conventional plastics. PHB is intracellularly accumulated by prokaryotic organisms. For the cells PHB functions manly as carbon and energy source, but all possible functions of PHB are still not known. Synechocystis (cyanobacteria) accumulates PHB using light as energy and CO2 as carbon source. The main trigger for PHB accumulation in cyanobacteria is nitrogen and phosphorous depletion with simultaneous surplus of carbon and energy. For the above reasons, obtaining knowledge about external factors influencing PHB accumulation is of highest interest. This study compares the effect of continuous light exposure and day/night (16/8 h) cycles on selected physiology parameters of three Synechocystis strains. We show that continuous illumination at moderate light intensities leads to an increased PHB accumulation in Synechocystis salina CCALA 192 (max. 14.2% CDW - cell dry weight) compared to day/night cycles (3.7% CDW). In addition to PHB content, glycogen and cell size increased, while cell density and cell viability decreased. The results offer new approaches for further studies to gain deeper insights into the role of PHB in cyanobacteria to obtain bioplastics in a more sustainable and environmentally friendly way.

The Arabidopsis exocyst complex is involved in cytokinesis and cell plate maturation.

Cell reproduction is a complex process involving whole cell structures and machineries in space and time, resulting in regulated distribution of endomembranes, organelles, and genomes between daughter cells. Secretory pathways supported by the activity of the Golgi apparatus play a crucial role in cytokinesis in plants. From the onset of phragmoplast initiation to the maturation of the cell plate, delivery of secretory vesicles is necessary to sustain successful daughter cell separation. Tethering of secretory vesicles at the plasma membrane is mediated by the evolutionarily conserved octameric exocyst complex. Using proteomic and cytologic approaches, we show that EXO84b is a subunit of the plant exocyst. Arabidopsis thaliana mutants for EXO84b are severely dwarfed and have compromised leaf epidermal cell and guard cell division. During cytokinesis, green fluorescent protein-tagged exocyst subunits SEC6, SEC8, SEC15b, EXO70A1, and EXO84b exhibit distinctive localization maxima at cell plate initiation and cell plate maturation, stages with a high demand for vesicle fusion. Finally, we present data indicating a defect in cell plate assembly in the exo70A1 mutant. We conclude that the exocyst complex is involved in secretory processes during cytokinesis in Arabidopsis cells, notably in cell plate initiation, cell plate maturation, and formation of new primary cell wall.

Description and phylogeny of Zelenkaia trichopterae gen. et sp. nov. (Microsporidia), an aquatic microsporidian parasite of caddisflies (Trichoptera) forming spore doublets.

Two novel microsporidia infecting the fat body tissues in larvae of two hosts, Halesus digitatus and Micropterna sequax (Trichoptera, Limnephilidae), were investigated using light and electron microscopy and rDNA sequence analyses. The molecular and morphological characters of these isolates warrant creation of a new microsporidian genus, Zelenkaia gen. n., with two species, one named herein. Developmental stages of Zelenkaia spp. have single nuclei. In sporogony, a plasmodium with four nuclei gives rise by rosette-like budding to two pairs of uninucleate sporoblasts, each within a thin-walled, subpersistent sporophorous vesicle. Sporoblasts and mature spores adhere temporary together, forming doublets oriented in parallel, within the sporophorous vesicle. Spores are long-oval and uninucleate, and those of the type species Z. trichopterae measure 10.3×3.5µm and have 24-25 polar filament coils. Phylogenetic analysis based on rDNA places Zelenkaia spp. within the aquatic clade of microsporidia and, more specifically, in the clade containing some microporidia from amphipod hosts.

Cytocomposition of the vitellarium in Khawia sinensis Hsü, 1935 (Cestoda, Caryophyllidea, Lytocestidae): another caryophyllidean species with lamellar bodies and lipids.

The vitellarium of the invasive caryophyllidean tapeworm Khawia sinensis Hsü, 1935 from carp Cyprinus carpio L. was examined by means of transmission electron microscopy and cytochemical staining for glycogen with periodic acid-thiosemicarbazide-silver proteinate (PA-TSC-SP). A vitellarium consists of numerous follicles of irregular size that are interconnected by a net of vitelline ducts. Vitelline follicles are composed of vitelline cells at various stages of development that are interconnected by interstitial tissue. Vitelline follicles are surrounded by a cytoplasmic sheath associated with an intercellular matrix. Extensive development of the granular endoplasmic reticulum and Golgi complexes are both involved in the production of shell globules/shell globule clusters and characterise cytodifferentiation of vitellocytes. Nuclear and nucleolar transformation lead to the formation and storage of intranuclear glycogen, a feature specific for the Caryophyllidea. Newly observed within the mature vitellocytes of Khawia sp. is the presence of lamellar bodies and a few lipid droplets. These cytoplasmic inclusions first occur in the mature cells within the follicles and persist in the vitelline cells within vitelloducts and intrauterine eggs. Two types of lamellar bodies are detected: regular lamellar-structured body and irregular lamellar-structured body. None of the lamellar bodies are membrane bound. Results of the present study indicate that the formation of lamellar bodies may be closely related to the endoplasmic reticulum or shell globule clusters. Some of the shell globule clusters are transformed into lamellar body clusters. Ultrastructural features of vitellocytes in K. sinensis are compared with those of other monopleuroid, polypleuroid, and strobilated cestodes.

Microscopic morphology and distribution of the antennal sensilla in the double-spined bark beetle, Ips duplicatus (Coleoptera: Curculionidae).

The double-spined spruce bark beetle, Ips duplicatus, has become an infamous secondary pest of Norway spruce, causing extensive ecological and economic destruction in many Central European countries. Antennae are the primary olfactory organs that play a fundamental role in insect-host chemical communication; therefore, understanding morphology is crucial before conducting electrophysiological investigations. Here, we present our analysis of sensilla types on the antennal surface of I. duplicatus for the first time, using high-resolution-scanning electron microscopy. We studied the external morphological characteristics of antennae and the types, numbers, and distribution of the antennal sensilla in males and females. Our results revealed the presence of five different types of morphologically distinct sensilla: sensilla chaetica, sensilla basiconica, sensilla trichodea, sensilla coeloconica, and Böhm's sensilla. We observed two subtypes of sensilla chaetica (SChI and SChII), four subtypes of sensilla basiconica (SBI, SBII, SBIII, and SBIV), three subtypes of sensilla trichodea (STrII, STrIII, and STrIV) and two subtypes of sensilla coeloconica (SCoI and SCoII), respectively in I. duplicatus males and females. Minor differences in length and numbers between the sexes for some sensilla types were found. Distribution maps for different sensillar types were constructed, and specific areas for the respective sensilla were found. Possible functions of observed sensilla types are discussed. The present study provides a basis for future electrophysiological studies to understand how I. duplicatus detects ecologically important olfactory cues. RESEARCH HIGHLIGHTS: • The first report of morphology and distribution pattern of the antennal sensilla in Ips duplicatus is discussed. • A total of 6 main types and 11 antennal sensilla subtypes were observed in male and female Ips duplicatus. • Minor sex-specific differences were seen in the length and numbers in several sensilla types.

Selective and reversible disruption of mitochondrial inner membrane protein complexes by lipophilic cations.

Triphenylphosphonium (TPP) derivatives are commonly used to target chemical into mitochondria. We show that alkyl-TPP cause reversible, dose- and hydrophobicity-dependent alterations of mitochondrial morphology and function and a selective decrease of mitochondrial inner membrane proteins including subunits of the respiratory chain complexes, as well as components of the mitochondrial calcium uniporter complex. The treatment with alkyl-TPP resulted in the cleavage of the pro-fusion and cristae organisation regulator Optic atrophy-1. The structural and functional effects of alkyl-TPP were found to be reversible and not merely due to loss of membrane potential. A similar effect was observed with the mitochondria-targeted antioxidant MitoQ.