Body musculature of Beauchampiella eudactylota (Gosse, 1886) (Rotifera: Euchlanidae) with additional new data on its trophi and overall morphology

This study presents the results of confocal laser scanning microscopy and fluorescence‐labelled phalloidin used to visualize the system of body musculature in Beauchampiella eudactylota. Moreover, the poorly known trophi of B. eudactylota are described based on scanning electron microscopy. In total, four paired longitudinal muscles (musculi longitudinales I–IV) and three circular muscles (musculi circulares I–III) were identified. Among these are the musculus longitudinalis ventralis, the musculus longitudinalis dorsalis and the musculus circumpedalis as documented in previous studies for other rotifer species. Compared to other species, B. eudactylota is characterized by the low number of lateral longitudinal muscles and the absence of some longitudinal muscles (musculi longitudinales capitum) and circular muscles (corona sphincter, musculus pars coronalis). Moreover, scanning electron microscopic data on the trophi of B. eudactylota reveal a number of striking similarities to the trophi in some species of Epiphanidae. This suggests that either (1) these similarities represent plesiomorphic characters present both in Epiphanidae and B. eudactylota or (2) they are synapomorphic features of B. eudactylota and some species of Epiphanidae, which would question the monophyly of Euchlanidae.     

In vitro excystation of Echinostoma paraensei (Digenea: Echinostomatidae) metacercariae assessed by light microscopy,morphometry and confocal laser scanning microscopy

Trypsin and bile salts have been identified as important triggers for excystation of Echinostoma metacercariae. Although excystation in trematodes is a well-known phenomenon, some morphological developmental changes remain to be elucidated. In order to gain further insight into the in vitro development of metacercariae, we assayed different cultivating conditions: 0.5% trypsin and 0.5% bile salts; 1% trypsin and 1% bile salts; 1% trypsin and 0.5% bile salts; 0.5% bile salts; or 0.5% trypsin. By means of light microscopy and confocal microscopy, we characterized each encysted, activated, breached and excysted stage based on the morphological features. However, breached and excysted stages were not revealed in both bile salts and trypsin-free medium. Excretory concretions (25 ± 3.9) were visualized within excretory tubules, close to the ventral sucker and genital anlage. The oral sucker armed with spines and digestive system was similar to those of adult worms. The reproductive system is composed of a genital anlage and the cirrus sac primordium. In short, trypsin and bile salts associated were fundamental for the in vitro metacercariae excystation of Echinostoma paraensei. This article presents the first detailed information of all stages of metacercariae excystation obtained through light and confocal microscopy.     

Structural analysis of microparticles by confocal laser scanning microscopy

This study demonstrates the potential of conforcal laser scanning microscopy (CLSM) as a characterization tool for different types of microparticles. Microparticles were prepared by various methods including complex coacervation, spray drying, double emulsion solvent evaporation technique, and ionotropic gelation. Protein drugs and particle wall polymers were covalently labeled with a fluorescent marker prior to particle preparation, while low molecular weight drugs were labeled by mixing with a fluorescent marker of similar solubility properties. As was demonstrated in several examples, CLSM allowed visualization of the polymeric particle wall composition and detection of heterogeneous polymer distribution or changes in polymer matrix composition under the influence of the drug. Furthermore, CLSM provides a method for three-dimensional reconstruction and image analysis of the microparticles by imaging several coplanar sections throughout the object. In conclusion, CLSM allows the inspection of internal particle structures without prior sample destruction. It can be used to localize the encapsulated compounds and to detect special structural details of the particle wall composition.     

Combining confocal laser scanning and transmission electron microscopy for revealing the mastax musculature in Bryceella stylata (Milne, 1886) (Rotifera: Monogononta)

The rotiferan jaw apparatus (mastax) is characterized by enormous plasticity and according to morphology and feeding strategy, different mastax types can be distinguished. The cuticular hard parts (trophi) of the mastax are often highly specialized and have both a major taxonomic and phylogenetic relevance. Owing to numerous light and scanning electron microscopic studies, the morphology of the trophi is well known but only few attempts have been made to analyze the morphology and functionality of the mastax as a whole. Particularly, the complex muscular system connecting the individual trophi elements and moving them against each other was disregarded in the past. Therefore, the subject of the present study is a detailed analysis of the mastax musculature of the proalid rotifer Bryceella stylata using a combination of transmission electron and confocal laser scanning microscopic techniques, previously applied for revealing the somatic musculature in rotifers exclusively. Based on ultrathin serial sections and phalloidin-dyed specimens, a total number of six paired and two unpaired individual mastax muscles have been identified for the modified malleate trophi system of B. stylata. Possibly homologous muscles in other, so far investigated rotifer species are discussed as well as functional considerations of the individual mastax muscles and their interaction when moving the trophi elements are suggested.     

Cellular localization of a pollen-specific mRNA by in situ hybridization and confocal laser scanning microscopy

Summary The application of confocal laser scanning microscopy together with in situ hybridization experiments in tobacco pollen enabled a detailed localization of a pollen-specific mRNA. The three-dimensional distribution of this specific mRNA over the whole pollen grain was reconstructed by means of optical sections of one specimen.     

The transport kinetics of lanthanide species in a single erythrocyte probed by confocal laser scanning microscopy

 A novel method has been developed to visualize and follow the temporal course of lanthanide transport across the membrane into a single living erythrocyte. By means of confocal scanning microscopy and the optical section technique, the entry of lanthanide ions was followed by the fluorescence quenching of fluorescein isothiocyanate (FITC)-labeled membrane and cytosol. From the difference of the quenching kinetics of the whole section and the central area, the time for diffusion through the membrane and the diffusion in the extracellular and intracellular media can be deduced. To clarify the mechanism of lanthanide-induced fluorescence quenching of FITC-labeled erythrocytes and to ensure that this reaction can be used in this method, the reaction was investigated by steady-state fluorescence techniques. The results showed that the lanthanides strongly quenched the florescence emitted by FITC covalently bound to membrane proteins and cytosolic proteins. The static quenching mechanism is responsible for the fluorescence quenching of FITC-labeled proteins by Ln species. The quenching mechanism is discussed on the basis of complex formation. The dependence of fluorescence quenching on both ion size and the total orbital angular momentum L supports the complexation mechanism. The transport time across the membrane is strikingly correlated with Ln species and extracellular concentration. For a given concentration, the transport time of [Ln(cit)₂]^3– is much shorter than that of Ln³⁺, since they enter the cells via the anion channel. This is supported by the inhibition effect of 4,4′-diisothiocyanato-2,2′-stilbenendisulfonate on the transport of [Ln(cit)₂]^3–. On the other hand, the transport of free Ln³⁺ might be attributed to the enhanced permeability of erythrocytes owing to Ln³⁺ binding. These findings strongly demonstrate the existence of the non-internalization mechanism of Ln species uptake by erythrocytes. Received: 7 January 1999 / Accepted: 7 May 1999     

Confocal laser scanning microscopy as an analytical tool in chromatographic research

In recent years, confocal laser scanning microscopy has been developed into a non-invasive tool to probe intra-particle profiles of protein in chromatographic adsorbents. A necessary prerequisite when using this technique lies in the labeling of proteins with fluorescent probes. The quality of the obtained results is thus strongly dependent on the probes used, its sensitivity on experimental parameters and the change of protein characteristics upon binding. In this review, the fundamental issues when using fluorescent probes are described, before giving a critical evaluation on published literature in the field of confocal laser scanning microscopy for the analysis of chromatographic principles.     

四唑盐减低法结合激光共聚焦显微镜定量分析表皮葡萄球菌生物被膜体外模型

目的建立体外表皮葡萄球菌(Staphylococcus epidermidis)生物膜(biofilm,BF)模型,观察和定量分析表皮葡萄球菌BF的动态形成过程。方法采用可形成BF的表皮葡萄球菌RP62A,平板法建立体外BF模型,四唑盐(tetrazolium salt,XTT)减低法定量检测BF形成过程中细菌活力的变化,激光共聚焦显微镜(confocal laser scan-ning microscopy,CLSM)结合图像结构分析软件(image structure analyer,ISA)对BF形成过程结构参数进行动态分析,扫描电镜(scanning electron microscope,SEM)观察BF形成过程中的形态结构。结果在12、24和48 h时,XTT减低法A450的值分别为2.39±0.48、3.41±0.18和3.92±0.27,P0.05;ISA软件定量分析显示在区域孔径(AP)的值分别为0.84±0.08、0.68±0.01和0.59±0.13,P0.05,平均扩散距离(ADD)的值分别为1.34±0.24、1.49±0.09和1.89±0.39,P0.05,结构熵(TE)的值分别为4.71±0.82、8.69±0.68和8.94±0.28,24 h、48 h与12 h相比,P0.05。结论表皮葡萄球菌BF的形成是个动态的过程,24 h时BF基本形成,48 h BF结构更加复杂。XTT减低法,CLSM结合ISA软件,SEM三种方法联合使用是观察和定量分析体外BF模型较理想的方法。     

DNA-fluorescence of mammalian intra-nucleolar chromatin detected by confocal laser scanning microscopy (CLSM)

The complex spatial DNA distribution in the mammalian interphase nucleus was investigated in Feulgen stained thick sections through mouse trophoblast giant nuclei after Lowicryl embedding. DNA-fluorescence was visualized using confocal laser scanning microscopy. Our results show that the spatial arrangement of major interphase chromatin areas can be precisely documented, including the distribution of small intra-nucleolar chromatin zones.     

Localisation of fungal hyphae in wood using immunofluorescence labelling and confocal laser scanning microscopy

This study explored the feasibility of using immunofluorescence labelling in conjunction with confocal laser scanning microscopy (CLSM) for detection of common fungal colonisers of unseasoned radiata pine in New Zealand. Wood sections infected with Ophiostoma piceae were treated with monoclonal antibody IF3 (1), and then Oregon green 514 goat anti-mouse IgG, a fluorescent secondary antibody. Additional wood sections infected with other Ophiostoma spp., Sphaeropsis sapinea, Leptographium procerum, Trichoderma sp. and Phlebiopsis gigantea were treated similarly to determine whether the antibody was specific to O. piceae or was recognising other fungal species. Sections were examined using phase contrast and fluorescence light microscopy prior to CLSM. Immunolabelled fungal hyphae showed relatively weak fluorescence compared to the strong autofluorescence of wood cell walls and extractives. Labelled hyphae of O. piceae were detected in wood using CLSM but not with ordinary fluorescence microscopy. This is because CLSM has stronger illumination power and superior imaging ability compared with ordinary fluorescence microscopy. The monoclonal antibody did not cross-react with the other Ophiostoma species. However, non-specific antibody binding was observed with L. procerum and Trichoderma species. Furthermore, cell walls of L. procerum showed strong autofluorescence with optical properties similar to wood extractives when examined prior to incubation with the monoclonal and secondary antibody, therefore cross-reactivity tests were inconclusive for Leptographium and Trichoderma species. The current investigation demonstrated that CLSM provides possibilities for future investigations on in situ interactions of common radiata pine fungal colonisers, with one another and with wood.     

Development of the reproductive system of Echinostoma paraensei in Mesocricetus auratus analyzed by light and confocal scanning laser microscopy

This study was performed to gain insight into the maturation of the reproductive system of Echinostoma paraensei worms grown in an early infection of Mesocricetus auratus. Hamsters were infected with 100 metacercariae and necropsied on days 3, 5, 7, 10 and 14 post infection (dpi). Recovered flukes stained with hydrochloric carmine were preserved as whole mounts and analyzed by light and confocal scanning laser microscopy. The average worm recovery was 43.7 per host. Images of the male and female reproductive systems were taken. The ovary and anterior and posterior testis were evidenced on day 3, while the ootype and cirrus sac were present on day 5. Confocal imaging showed primordium testis and ovary as a cluster of primordial cells from day 3 onward. The testes, ovary, cirrus sac and uterus organs were already present during the first week of life. The two testes were seen as individual structures on 7 dpi while the cirrus sac and vitelline glands were in development. The ovary was connected to the uterus while the ootype was adjacent to it. Both testes were larger than the ovary, showing cells at different stages of development, but with few bundles of functional spermatozoa in 10 day-old worms. On day 14, eggs and spermatozoa were seen in the uterus and seminal vesicle, respectively, while oocytes appeared in the ootype as fertilized eggs. We conclude that the reproductive system of E. paraensei was functional on 14 dpi in the hamsters.     

Mode of infection of the corn earworm (Heliothis zea) by Beauveria bassiana as revealed by scanning electron microscopy

Conidia from highly pathogenic mutants of Beauveria bassiana germinate quickly (within 18 hr) on the surface of corn earworm larvae (Heliothis zea) and immediately begin penetration of the cuticle. Enzymes produced by the penetrating hyphae degrade the cuticle since holes are formed at the point of entry. Clustering of conidia around nodules of larvae is often seen, but penetration is not restricted to such areas. Direct evidence is presented to show that conidia can also germinate inside of spiracle openings and could invade larvae by this route. Once inside the hemocoel, the fungus multiplies extensively; however, larval death occurs with only minimal breakdown of internal tissues. During mummification, outgrowths of fungal hyphae occur first and most extensively in the intersegmental regions of larvae. Conidia from mutants exhibiting low levels of pathogenicity are either significantly delayed or do not germinate on larval surfaces. When germination does occur, hyphae from such mutants do not penetrate immediately; instead, extensive surface hyphal growth, with or without eventual penetration of the integument, is evident.     

Comparative morphology of the somatic musculature in species of Hexarthra and Polyarthra (Rotifera, Monogononta): Its function in appendage movement and escape behavior

Species of Hexarthra and Polyarthra are freshwater rotifers with well-known escape behaviors that result from interactions with planktonic predators. Both rotifers bear a suite of mobile appendages that function in evasive maneuvers and saltatory jumps through the water column, but the anatomical and functional bases of these actions are poorly understood. Here, we use a combination of phalloidin staining, confocal laser scanning microscopy, and video analysis to describe the morphology of the somatic muscles that supply the mobile appendages in order to understand how they function in escape behavior. Results show that species of Hexarthra, which bear six radially distributed limbs, possess a highly complex trunk musculature that supplies the inside of each limb with its own abductor and adductor muscles, i.e., a direct muscle supply. The singular dorsal and ventral limbs each receive a pair of large abductor and adductor muscles (four muscles total per limb), while the paired dorsolateral and ventrolateral limbs each receives three muscles (two abductors, one adductor per limb). Contraction of the abductor muscles creates a power stroke in the form of an anterior sweep of the limbs, which leads to a three-dimensional tumbling of the rotifer through the water column. Alternatively, species of Polyarthra possess 12 blade-like appendages that are arranged into four equal bundles; each bundle receives an indirect muscle supply that attaches to the shoulder of the paddles. A single longitudinal paddle muscle supplies each dorsolateral bundle, while a pair of longitudinal paddle muscles supplies each ventrolateral bundle. Contraction of these muscles, whether singly or in concert, functions to abduct the paddles in a power stroke, leading to rotation of the body and movement of the rotifer. The recovery stroke is hypothesized to be a multi-step process that begins with reorientation of the appendages prior to adduction, followed by contraction of various muscles to antagonize the paddle muscles. In total, these observations reveal novel complexities in the rotifer muscular system that aids our understanding of the biophysics of predator avoidance in appendage-bearing rotifers.     

Confocal laser scanning microscopy of fluorescently stained wood cells: a new method for three-dimensional imaging of xylem elements

Summary Xylem cells were fluorescently stained with periodic acid — Schiff reaction or with Schiffs reagent alone and studied by confocal laser scanning microscopy. Single images with extremely low depth of focus, series of optical sections, computed stereo scopic images and shadow casting images as well as x-z images are obtained. In contrast to scanning electron microscopy, not only are the surfaces imaged, but elements concealed by other structures can be visualized by this system. Quantitative data on cell depth are provided and differences in lignification are detected.     

Comparative studies on scanning electron microscopy of trophi of the genus Filinia Bory De St. Vincent (Rotifera)

Trophi of Filinia species from 16 South Island lakes and three North Island lakes of New Zealand were examined and compared with specimens from Australia, Austria, Belgium, and Turkey. Five species of Filinia (brachiata, longiseta, cf. pejleri, novaezealandiae, and terminalis) were positively identified from the New Zealand samples. Numbers of unci teeth are considered to be the most reliable features for identification within the genus. Numbers obtained from SEM for other species of Filinia (australiensis, grandis, and hofmanni) are also listed for the first time.     

Musculature in three species of <Emphasis Type="Italic">Proales</Emphasis> (Monogononta,Rotifera) stained with phalloidin-labeled fluorescent dye

The musculature in the rotifer species Proales daphnicola, P. reinhardti and P. fallaciosa was stained with phalloidin-labeled fluorescent dye and compared using confocal laser scanning microscopy. All three species share several homologous muscle systems, but each systems detailed morphology varies among the species both concerning appearance, number and location. The obtained results were compared with data from other rotifers and it was concluded that the muscles pars coronalis and the corona sphincter probably represent conditions in Ploima or Monogononta, while incomplete circular muscles and dorsal and ventral trunk retractors might be part of the eurotatorian ground pattern.     

Development of Schistosoma mansoni in the laboratory rat analyzed by light and confocal laser scanning microscopy

The kinetic of maturation (schistogram) of Schistosoma mansoni worms grown in laboratory rats was studied by light and confocal laser scanning microscopy. Infected rats with the BH strain were weekly euthanized 3-9 weeks pi. Recovered flukes stained with hydrochloric carmine were preserved as whole-mounts and analyzed by confocal and brightfield microscopy. Worms displayed varying degrees of maturation of the reproductive system at weeks 3-6. Male worms showed complete maturation of the reproductive system at week 6, while female worms completed their maturation at week 7. Males presented few tubercles in tegument in all weeks. Despite the presence of a developing embryo within the ootype, no uterine egg was found. The schistogram in rats follows a pattern similar to that observed in mice hosts.