Morpho-histochemical characterization of salivary gland cells of males of the tick <Emphasis Type="Italic">Rhipicephalus</Emphasis><Emphasis Type="Italic">sanguineus</Emphasis> (Acari: Ixodidae) at different feeding stages: description of new cell types

This study describes the changes undergone by cells of the salivary glands of unfed and feeding (at day two and four post-attachment) Rhipicephalus sanguineus males, as well as new cell types. In unfed males, types I and II acini are observed with cells “undifferentiated”, undefined 1 and 2 (the latter, with atypical granules), a, c1 and c3; type III is composed of cells d and e; and type IV present cells g. In males at day two post-attachment, type I acini exhibit the same morphology of unfed individuals. An increase in size is observed in types II, III, and IV, as cells are filled with secretion granules. Some granules are still undergoing maturation. In type II acinus, cells a, b and c1–c8 are observed. Cells c7 and c8 are described for the first time. Cells c7 are termed as such due to the addition of polysaccharides in the composition of the secretion granules (in unfed individuals, they are termed undefined 1). Type III acini exhibit cells d and e completely filled with granules, and in type IV, cells g contain granules in several stages of maturation. In males at day four post-attachment, type I acini do not exhibit changes. Granular acini exhibit cells with fewer secretion granules, which are already mature. In type II acini, cells a, b, c1–c5 are present, type III exhibit cells d and e, and type IV contain cells g with little or no secretion. This study shows that in the salivary glands of R. sanguineus males, cells a, c1, and c3 of type II acinus, and cells d and e of type III do not exhibit changes in granular content, remaining continuously active during the entire feeding period. This indicates that during the intervals among feeding stages, gland cells reacquire the same characteristics found in unfed individuals, suggesting that they undergo reprogramming to be active in the next cycle.     

Ultrastructural changes in <Emphasis Type="Italic">Yarrowia lipolytica</Emphasis> cells under stress conditions

Ultrastructural organization of the aerobic yeast Yarrowia lipolytica was studied under conditions of oxidative, heat, and ethanol stresses. It was shown that the following uniform changes in cell ultrastructure did not depend on the type of stress: enlargement of mitochondria, enhanced number and enlargement of peroxisomes, and formation of lipid granules. Similar ultrastructural changes also occurred during the transition of cells to the stationary growth phase. It was shown for the first time that accumulation of polyphosphate granules occurred as a stress response in yeasts. Moreover, numerous globular structures of unknown nature appeared on the cell wall surface under oxidative or heat stress. Under ethanol stress, the cells developed clearly marked deep invaginations of the cytoplasmic membrane. (The same changes in the cytoplasmic membrane were observed in the cells grown on ethanol.) Variations of the cell envelope structure along with the formation of polyphosphate granules were not observed in the stationary growth phase. Ultrastructural changes in the cells under stress conditions are in agreement with the previous data on survival, respiratory activity, and variations of the antioxidant systems.     

Use of the multipurpose transposon Tn<Emphasis Type="Italic">KPK2</Emphasis> for the mutational analysis of chromosomal regions upstream and downstream of the<Emphasis Type="Italic"> sipF</Emphasis> gene in<Emphasis Type="Italic"> Bradyrhizobium japonicum</Emphasis>

The DNA regions upstream and downstream of the Bradyrhizobium japonicum gene sipF were cloned by in vivo techniques and subsequently sequenced. In order to study the function of the predicted genes, a new transposon for in vitro mutagenesis, TnKPK2, was constructed. This mutagenesis system has a number of advantages over other transposons. TnKPK2 itself has no transposase gene, making transposition events stable. Extremely short inverted repeats minimize the length of the transposable element and facilitate the determination of the nucleotide sequence of the flanking regions. Since the transposable element carries a promoterless phoA reporter gene, the appearance of functional PhoA fusion proteins indicates that TnKPK2 has inserted in a gene encoding a periplasmic or secreted protein. Although such events are extremely rare, because the transposon has to insert in-frame, in the correct orientation, and at an appropriate location in the target molecule, a direct screening procedure on agar indicator plates permits the identification of candidate clones from large numbers of colonies. In this study, TnKPK2 was used for the construction of various symbiotic mutants of B. japonicum. One of the mutant strains, A2-10, which is defective in a gene encoding a protein that comigrates with bacterioferritin (bcpB), was found to induce the formation of small and ineffective nodules.Communicated by A. Kondorosi     

Interplay of transposon-silencing genes in the germline of <Emphasis Type="Italic">Drosophila melanogaster</Emphasis>

Complexes of Piwi family proteins with short piRNAs (Piwi-interacting RNAs) are responsible for silencing transposable elements in animal reproductive organs. In Drosophila melanogaster, three proteins (Piwi, Aub, and Ago3) are members of the Piwi family. Piwi is the nuclear protein of somatic and germinal ovarian cells, whereas Aub and Ago3 are cytoplasmic proteins involved in piRNA amplification in perinuclear granules that constitute special organelles of germinal cells called nuage. Mutations in genes of the piRNA system are known to cause derepression of several transposable elements. In this study, we compared quantitatively changes in expression of a larger number of elements in the case of mutations in the piwi gene, genes aub, mael, and spn-E, which encode proteins of nuage granules, and armi gene coding an RNA helicase, the lack of which does not interfere with cytoplasmic piRNA amplification but disturbs nuclear localization of Piwi protein. We found that the genes piwi, armi, aub, spn-E, and mael interact to induce silencing of some retrotransposons (HMS-Beagle, Gate and HeT-A); the same genes, except piwi, are involved in repression of I and G elements. We propose that Armi is involved in control of not only nuclear Piwi localization. Our data suggest the relation of nuage proteins Aub, Spn-E, and Mael to Piwi-mediated silencing of retrotransposons Gate and HMS-Beagle in the nucleus. In general, our results corroborate the idea of genome stabilization by means of various silencing strategies specific to different transposable elements. At the same time, our data suggest the existence of yet unknown mechanisms of interplay between nuclear and cytoplasmic components of the piRNA machinery in germinal cells.     

Ein Mechanismus zur Steuerung des Lichtflusses in den Rhabdomeren des Komplexauges von Musca

Summary In the ommatidia of Musca, the light flux transmitted by each one of the rhabdomeres of sense cells no. 1 to 6 decreases as a function of time if light falls onto these rhabdomeres. With a similar time course the light flux reflected from these rhabdomeres increases. These changes take place within a few seconds following illumination. The results have been established in the intact animal using changes in the appearance of the pseudopupil as indicator and also in surviving preparations of the eye with direct inspection of the rhabdomeres.The changes are interpreted as a consequence of interactions between pigment granules in the sense cells and electromagnetic fields induced outside the rhabdomeres by light travelling on the inside: In the dark adapted situation the granules are quite distant from the rhabdomeres, the interaction is negligible. During light adaptation the granules move close to the rhabdomeres, and as a consequence, total reflection of the light in the rhabdomere is frustrated. The relatively rapid changes in the optical characteristics of the rhabdomeres are explained by the fact that the distance, the granules have to move in order to switch from one condition to the other is in principle on the order of the wavelength of light.The results indicate, that the changes in the position of the granules are induced by the excitation of the respective sense cells themselves, for instance by the degree of their depolarisation. No interaction between the sense cells of one ommatidium nor between those of different ommatidia could be found.The function of the movement of the pigment granules is interpreted as a means to protect the sense cells no. 1 to 6 against strong illumination. — Movement of pigment granules is not induced in sense cells no. 7 and 8 with light intensities which give maximal response in sense cells no. 1 to 6.

---

---

Wertvolle Diskussionen verdanken wir Herrn Dr. K. G. Götz sowie Herrn Prof. W. Reichardt. Wir danken Fräulein T. Wiegand für Mithilfe bei den Experimenten sowie Herrn E. Freiberg für das Fertigstellen der Abbildungen.     

A transferable heterogeneous two-hybrid system in <Emphasis Type="Italic">Escherichia coli</Emphasis> based on polyhydroxyalkanoates synthesis regulatory protein PhaR

Background  

Polyhydroxyalkanoate (PHA) synthesis regulatory protein PhaR contains a DNA binding domain (DBD) and a PHA granule binding domain (GBD), it anchors to the promoter region of PHA granule-associated protein (PhaP) to repress phaP expression. However, PhaR will bind to PHB granules and be released from phaP promoter region when PHA granules are formed in vivo, initiating expression of phaP gene. Based on this regulatory mechanism, a bacterial two-hybrid system was developed: PhaR was separated into two parts: DBD was used to fuse with the bait, GBD with the prey, and phaP was replaced by a reporter gene lacZ. However, GBD protein expressed in vivo formed inclusion bodies. Thus, PhaP with strong binding ability to PHB granules was employed to replace GBD.     

A secondary effect of transformation in Rhizobium leguminosarum transgenic for Bacillus thuringiensis subspecies tenebrionisδ-endotoxin (cryIIIA) genes

 By introducing Bacillus thuringiensis subspecies tenebrionisδ-endotoxin genes (cryIIIA) into Rhizobium leguminosarum we have produced strains for the biological control of Sitona larvae. Comparisons between a transgenic and the parent strain show that transformation has induced changes not associated with the intended function of the transgene. Although growth rates in laboratory cultures are similar for both strains, the ability to compete for nodule occupancy is greater in the transgenic than in the non-transformed parent strain. This result demonstrates the importance of studying ecological and agronomic characters of transgenic micro-organisms that could have a bearing on the safety and success of their release into the environment, even if they are not thought to be connected with the transgenes introduced. Received: 20 April 1997 / Accepted: 2 June 1997     

Micromorphological differences between some European and American <Emphasis Type="Italic">Fraxinus</Emphasis> (<Emphasis Type="Italic">Oleaceae</Emphasis>) species

Micromorphological differences in leaves and pollen between two American (Fraxinus americana L., F. pennsylvanica Marshall) and two European (F. angustifolia Vahl, F. excelsior L.) ash species were studied using scanning electron microscope. The types, dimensions and distribution of characteristic trichomes were established and measured. Capitate hairs on the leaves had the same shape in all researched ash species. Acicular hairs were regularly present in two American ash species, but very rarely in the glabrous phase of F. angustifolia and F. excelsior. Only F. americana had coronulate abaxial surface of leaves. Pollen of F. angustifolia and F. excelsior had 3 (tricolpate) apertures, and F. americana and F. pennsylvanica 4 (stephanocolpate) apertures. Based on the appearance of the reticulum it’s possible to clearly distinguish all four species. F. angustifolia and F. pennsylvanica had muri with transversal ridges and seldom granules. Muri of F. excelsior and F. americana had slightly visible transversal ridges, and because of that noticeable granules.     

Single-culture aerobic granules with <Emphasis Type="Italic">Acinetobacter calcoaceticus</Emphasis>

Aerobic granules are cultivated by a single bacterial strain, Acinetobacter calcoaceticus, in a sequencing batch reactor (SBR). This strain presents as a good phenol reducer and an efficient auto coagulator in the presence of phenol, mediated by heat-sensitive adhesins proteins. Stable 2.3-mm granules were formed in the SBR following a 7-week cultivation. These granules exhibit excellent settling attributes and degrade phenol efficiently at concentrations of 250–2,000 mg l⁻¹. The corresponding phenol degradation rate reached 993.6 mg phenol g⁻¹ volatile suspended solids (VSS) day⁻¹ at 250 mg l⁻¹ phenol and 519.3 mg phenol g⁻¹ VSS day⁻¹ at 2,000 mg l⁻¹ phenol concentration. Meanwhile, free A. calcoaceticus cells were fully inhibited at phenol >1,500 mg l⁻¹. Denaturing gradient gel electrophoresis fingerprint profile demonstrated no genetic modification in the strain during aerobic granulation. The present single-strain granules showed long-term structural stability and performed high phenol degrading capacity and high phenol tolerance. The confocal laser scanning microscopic test revealed that live A. calcoaceticus cells principally distributed at 200–250 μm beneath the outer surface, with an extracellular polymeric substance layer covering them to defend phenol toxicity. Autoaggregation assay tests demonstrated the possibly significant role of secreted proteins on the formation of single-culture A. calcoaceticus granules.     

Starter culture of <Emphasis Type="Italic">Pseudomonas veronii</Emphasis> strain B for aerobic granulation

Aggregation of bacterial cells is used in formation of microbial granules. Aerobically grown microbial granules can be used as the bio-agents in the treatment of wastewater. However, there are problems with start up of microbial granulation and biosafety of this process. Aim of this research was selection and testing of safe microbial strain with high cell aggregation ability to shorten period of microbial granules formation. Five bacterial strains with cell aggregation index higher than 50% have been isolated from the granules. Strain of Pseudomonas veronii species was considered as most probably safe starter culture for granulation because other strains belonged to the species known as human pathogens. The microbial granules were formed after 3 days of cultivation in case when P. veronii strain B was applied to start-up aerobic granulation process using model wastewater. The granules were produced from activated sludge after 9 days of cultivation. Microbial aggregates produced from starter culture of P. veronii strain B were more compact (sludge volume index was 70 ml/g) than those produced from activated sludge (sludge volume index was 106 ml/g). It is a first proof that application of selected safe starter pure culture with high cell aggregation ability can accelerate and enhance formation of microbial granules.     

Shortwave light filtration effect on spectral sensitivity of two shrimp populations of <Emphasis Type="Italic">M. relicta</Emphasis> (Mysida)

Microspectrophotometric measurements of screening granules in Mysis relicta eyes showed that most of the granules have xanthommatin spectra (7n_max 455 nm) with selective absorption of blue light. We calculated spectral sensitivity of M.relicta eyes using screening granules absorption spectra and visual pigment absorption spectra. According to our computations the calculated spectral sensitivity curve appears to be in a good correspondence with the real spectral sensitivity.     

Impact of Legume Flour Addition on Pasta Structure: Consequences on Its <Emphasis Type="Italic">In Vitro</Emphasis> Starch Digestibility

Pasta is popular for its ease of cooking and its low glycaemic index (GI). This interesting nutritional property can be attributed to its specific compact structure generally described as a protein network entrapping starch granules. Despite this low GI, pasta is poor in fibres and lack some essential amino acids. To enhance its nutritional composition, pasta can be fortified with non-traditional ingredients such as legume flours. The objective of this study was to investigate the impact of legume flour addition on pasta structure and the inherent consequences on the in vitro digestibility of starch. The addition of a high level (35%, w/w) of legume flour, especially split pea flour, induced some minor structural changes in pasta. The inclusion of fibres, the dilution of gluten proteins by albumins and globulins, and the larger amount of thin protein films (in split pea pasta) may have favoured higher susceptibility of starch to digestive enzymes. At the opposite, the presence of some partially gelatinised starch granules in the core of fortified pasta may have favoured the decrease in the in vitro starch digestibility. As a consequence, a high level of legume flour addition in pasta did not have any significant impact on its in vitro starch digestibility. A high level of split pea and faba bean flours can thus be added to pasta to increase its nutritional composition while keeping its low glycaemic index.     

Comparative ultrastructure of Langerhans‐like cells in spleens of ray‐finned fishes (Actinopterygii)

We studied the morphology and occurrence of splenic Langerhans‐like (LL) cells in species representing 11 orders of ray‐finned fishes, Actinopterygii. LL cells were frequent in spleen tissue of species among Cypriniformes, Esociformes, Salmoniformes, and Pleuronectiformes. These cells contained granules which resembled Birbeck granules known to occur in mammalian Langerhans cells. The ultrastructure of LL cells in Northern pike, Esox lucius, and in Atlantic halibut, Hippoglossus hippoglossus were similar to those reported in salmonids. LL cells found in cyprinids shared some characteristics with the LL cells in other Actinopterygii species, although unique structures distinguished them from the latter. They contained dense bodies within the Birbeck‐like (BL) granules, a characteristic that was never observed in species outside the Cypriniformes. Two types of BL granules were characterized in cyprinid LL cells. The ultrastructure of BL granules across the species is discussed. LL cells in all Actinopterygii species demonstrated close contacts with nearby cells, characterized by adherens‐like junctions. Additionally, multivesicular bodies were present within the cytoplasm and large aggregates of exosomes were observed closely associated with the plasma membrane suggesting their release from the cells. These structures are discussed in relation to mammalian dendritic cells. Macrophages found in European perch, Perca fluviatilis, blue gourami, Trichogaster trichopterus, and Atlantic halibut, Hippoglossus hippoglossus contained lysosomes and residual bodies with structures resembling Birbeck granules. These granules and cells were clearly distinct from LL cells. J. Morphol. 2010. © 2010 Wiley‐Liss, Inc.     

Licht- und elektronenmikroskopische Untersuchung des Corpus cardiacum der Eintagsfliege Ephemera danica Müll. (Ephemeroptera: Ephemeridae) während der Metamorphose*

Kaiser, H. 1980. Licht- und elektronenmikroskopische Untersuchung des Corpus cardiacum des Eintagsfliege Ephemera danica Müll. (Ephemeroptera: Ephemeridae) während der Metamorphose. [Light- and electron microscopic investigation on the corpus cardiacum in the mayfly Ephemera danica Müll. (Ephemeroptera: Ephemeridae) during metamorphosis.] (Zoologisches Institut der Universität Basel, Schweiz.) — Acta zool. (Stockh.) 61(2): 93–103. The corpus cardiacum (cc) of Ephemera danica is an unpaired organ in the dorsal wall of the aorta. During the last four instars there has been no significant change in the volume of the cc of the males. In the females, however, the cc enlarges in the final nymphal instar due to a thickening of the organ. Owing to a shortening prior to the subimaginal moult the volume has diminished in the subimagines and imagines. In the electron microscope different types of axons and secretory granules could be observed. The type a axons contain electron opaque granules with a mean diameter of 125 nm. The granules in the axons of type b are smaller (mean diameter 85 nm) and are more or less electron opaque, and the type c axons are filled with less electron opaque secretory droplets with mean diameters of 170 nm. In the fourth type of axons (d) the granules are approximately the same size as those of the type a but are more irregular in shape. In addition to the different axons two cell types—interstitial and secretory cells—are described, and the probable mechanism of the release of neurosecretory material is discussed. During metamorphosis only minor changes were apparent at the ultrastructural level.     

Encystment of <Emphasis Type="Italic">Azotobacter nigricans</Emphasis> grown diazotrophically on kerosene as sole carbon source

Encystment of Azotobacter nigricans was induced by its diazotrophic cultivation on kerosene. Its growth and nitrogenase activity were affected by kerosene in comparison to cultures grown on sucrose. Electron microscopy of vegetative cells showed that when nitrogenase activity was higher and the poly-β-hydroxybutyrate granules were not present to a significant extent, peripheral bodies were abundant. After 8 days of culture on kerosene, the presence of cysts with intracellular bunches of poly-β-hydroxybutyrate granules was observed. Germination of cysts bears germinating multicelled yet unbroken capsule cysts with up to three cells inside. This is the first report of encystment induction of Azotobacter species grown on kerosene.     

Effect of fungicides and insecticides applied during planting of sugarcane on viability of <Emphasis Type="Italic">Metarhizium anisopliae</Emphasis> and its efficacy against white grubs

The entomopathogenic fungus Metarhizium anisopliae (Metschnikoff) Sorokin is used for control of white grubs (Coleoptera: Scarabaeidae) in sugarcane fields in Queensland, Australia. Eight fungicides and three liquid insecticides are currently registered for application to sugarcane at planting, and may come into contact with M. anisopliae during its application from cane planters. Seven of these were tested for deleterious effects on two isolates of M. anisopliae, FI-147 and FI-1045, in laboratory and field experiments. In growth studies on medium, the four most commonly used fungicides were much more harmful to growth of M. anisopliae than the three insecticides. In a field experiment, where rice granules covered with spores of M. anisopliae were sprayed with each of four fungicides and two insecticides at very high rates and then covered with soil, only the fungicide Shirtan (methoxy ethyl mercuric chloride) showed any toxic effect on spore viability, with a reduction from 82 to 69%. No harmful effect of any chemical was detected in counts of colony-forming units in soil samples or in bioassays of treated soil using white grubs. Spore viability was not reduced when FI-1045 on rice granules (BioCane) was applied with fungicides through nine commercial planters, including five using Shirtan, compared with granules buried in untreated soil. Thus, we believe that M. anisopliae is compatible with these chemicals in commercial practice.     

Cloning and sequence analysis of the arginine deiminase gene from Mycoplasma arginini

Summary A deletion mutant of Rhizobium leguminosarum biovar viciae lacking the host-specific nodulation (nod) gene region (nodFEL nodMNT and nodO) but retaining the other nod genes (nodD nodABCIJ) was unable to nodulate peas or Vicia hirsuta, although it did induce root hair deformation. The mutant appeared to be blocked in its ability to induce infection threads and could be rescued for nodulation of V. hirsuta in mixed inoculation experiments with an exopolysaccharide deficient mutant (which is also Nod^–). The nodulation deficiency of the deletion mutant strain could be partially restored by plasmids carrying the nodFE, nodFEL or nodFELMNT genes but not by nodLMN. Surprisingly, the mutant strain could also be complemented with a plasmid that did not carry any of the nodFELMNT genes but which did carry the nodO gene on a 30 kb cloned region of DNA. Using appropriate mutations it was established that nodO is essential for nodulation in the absence of nodFE. Thus, either of two independent nod gene regions can complement the deletion mutant for nodulation of V. hirsuta. Similar observations were made for pea nodulation except that nodL was required in addition to nodO for nodulation in the absence of the nodFE genes. These observations show that nodulation can occur via either of two pathways encoded by non-homologous genes.Dedicated to the memory of the late Dr. David Goodchild     

The ultrastructure of guinea pig heterophil granulocytes and the heterogeneity of the granules

Summary The development of the heterophil granulocytes in the bone marrow of the guinea pig is described. During the maturation of these cells, three types of granule are formed, not only the azurophil and specific granules already described in other mammals but also a third type of granule referred to here as the nucleated granule. During the process of maturation of the cells, these three types of granule are formed successively. On this basis, two steps can be distinguished in the promyelocyte phase in which primary (nucleated and azurophil) granules are formed, i.e. an early and a late stage, nucleated granules being formed in early and azurophil granules in late promyelocytes. Secondary (specific) granules occur first in myelocytes. In mature heterophils of the guinea pig the granule population is composed of about 85% secondary granules, about 10% azurophil granules, and about 5% nucleated granules. The changes in the granule population during the maturation process were quantified. The observations and calculations point to the occurrence of three mitoses: one in the early and one in the late promyelocyte and the third in the myelocyte.