Silver Staining Method Research Articles

A Novel Deep Learning Approach for Analyzing Glomerular Basement Membrane Lesions in a Mouse Model of X-Linked Alport Syndrome

Alport syndrome is a rare kidney disease typically more severe in males due to its X-linked inheritance. However, female patients with heterozygous X-linked Alport syndrome (XLAS) can develop renal failure over time, necessitating accurate pathological assessment for effective therapy. A key pathological finding in female XLAS patients is the mosaic pattern of partial loss of α5 chains of type IV collagen. This study, using a mouse model of XLAS with a nonsense mutation (R471*) in the Col4a5 gene, analogous to human XLAS, aimed to examine the consistency of this pattern with the glomerular basement membrane (GBM) structure. A modified periodic acid-methenamine silver (PAMS) staining method was developed for clearer GBM visualization. The integrated images from COL4α5-stained fluorescence, PAMS, and low-vacuum scanning electron microscopy (LVSEM) into a single-slide section and applied supervised deep learning to predict GBM lesions. Results showed significant individual variability in urinary protein levels and histological lesions. Pathological parameters, including crescent formation, focal segmental glomerulosclerosis, and the COL4α5/α2 ratio, correlated with clinical parameters like urinary protein and plasma creatinine levels. Integrated LVSEM analysis revealed dense GBM regions corresponded to areas where COL4α5 was preserved, while coarse GBM (basket-weave lesions) occurred in COL4α5-deficient regions. These advanced techniques can enhance biopsy-based diagnosis of Alport syndrome and aid in developing AI diagnostic tools for diseases involving basement membrane lesions.

Anterograde and Retrograde Propagation of Inoculated Human Tau Fibrils and Tau Oligomers in a Non-Transgenic Rat Tauopathy Model.

The tauopathy of Alzheimer's disease (AD) is first observed in the brainstem and entorhinal cortex, spreading trans-synaptically along specific pathways to other brain regions with recognizable patterns. Tau propagation occurs retrogradely and anterogradely (trans-synaptically) along a given pathway and through exosomes and microglial cells. Some aspects of in vivo tau spreading have been replicated in transgenic mice models expressing a mutated human MAPT (tau) gene and in wild-type mice. In this study, we aimed to characterize the propagation of different forms of tau species in non-transgenic 3-4 months old wild-type rats after a single unilateral injection of human tau oligomers and tau fibrils into the medial entorhinal cortex (mEC). We determined whether different variants of the inoculated human tau protein, tau fibrils, and tau oligomers, would induce similar neurofibrillary changes and propagate in an AD-related pattern, and how tau-related pathological changes would correlate with presumed cognitive impairment. We injected human tau fibrils and tau oligomers stereotaxically into the mEC and examined the distribution of tau-related changes at 3 days and 4, 8, and 11 months post-injection using antibodies AT8 and MC1, which reveal early phosphorylation and aberrant conformation of tau, respectively, HT7, anti-synaptophysin, and the Gallyas silver staining method. Human tau oligomers and tau fibrils exhibited some similarities and some differences in their ability to seed and propagate tau-related changes. Both human tau fibrils and tau oligomers rapidly propagated from the mEC anterogradely into the hippocampus and various parts of the neocortex. However, using a human tau-specific HT7 antibody, 3 days post-injection we found inoculated human tau oligomers in the red nucleus, primary motor, and primary somatosensory cortex, a finding not seen in animals inoculated with human tau fibrils. In animals inoculated with human tau fibrils, 3 days post-injection the HT7 antibody showed fibrils in the pontine reticular nucleus, a finding explained only by uptake of human tau fibrils by incoming presynaptic fibers to the mEC and retrograde transport of inoculated human tau fibrils to the brainstem. Rats inoculated with human tau fibrils showed as early as 4 months after inoculation a spread of phosphorylated tau protein at the AT8 epitopes throughout the brain, dramatically faster propagation of neurofibrillary changes than with human tau oligomers. The overall severity of tau protein changes 4, 8, and 11 months after inoculation of human tau oligomers and tau fibrils correlated well with spatial working memory and cognition impairments, as measured by the T-maze spontaneous alternation, novel object recognition, and object location tests. We concluded that this non-trangenic rat model of tauopathy, especially when using human tau fibrils, demonstrates rapidly developing pathologic alterations in neurons, synapses, and identifiable pathways together with cognitive and behavioral changes, through the anterograde and retrograde spreading of neurofibrillary degeneration. Therefore, it represents a promising model for future experimental studies of primary and secondary tauopathies, especially AD.

The first case of Escherichia fergusonii with biofilm in China and literature review

BackgroundEscherichia fergusonii is a rare opportunistic pathogen in humans and animals, especially with biofilm.MethodsIn one case, E. fergusonii with biofilm was detected in the bile, and silver staining was used to prove it had biofilm. The clinical characteristics and drug susceptibility of eight cases of E. fergusonii retrieved from the literature were also summarized.ResultsThis is a case of E. fergusonii with biofilm, which has not been reported in China. The 8 cases retrieved from the literature did not specify whether they had biofilm, but we analyzed their clinical characteristics and drug susceptibility. All patients were treated with antimicrobial drugs. 8 cases showed sensitivity to piperacillin/tazobactam and imipenem in 6 cases (75%), but poor sensitivity to levofloxacin and ciprofloxacin.ConclusionThe silver staining method proved biofilm in this case, which is the first case of E. fergusonii with biofilm in China.

The taxonomy and molecular phylogeny of two epibiotic colonial peritrich ciliates (Ciliophora, Peritrichia)

Two colonial sessilid peritrichs, Epistylis qingdaoensis n. sp. and Carchesium cyclopidarum Nenninger, 1948, were isolated from a marine crustacean and a freshwater mayfly, respectively. Morphological characters for each species were revealed by in vivo observations and silver staining methods. Epistylis qingdaoensis n. sp. is characterized by the apperance of the colony which is up to 250 μm high and usually contains fewer than eight zooids, the single-layered peristomial lip, the conspicuously conical peristomial disc, and the structure of infundibular polykinety 3 which comprises three isometric ciliary rows. Carchesium cyclopidarum is recognized by the colony height of about 600 μm, the small zooid size of 35–50 × 20–30 μm, and the low number of silverlines. Phylogenetic analyses based on small subunit ribosomal DNA sequences were performed to reveal their evolutionary relationships. Surprisingly, neither species clustered with its congeners. Epistylis qingdaoensis n. sp. nested within a clade of Zoothamnium species that was sister to a clade comprising core Epistylis species and several Zoothamnium species. Carchesium cyclopidarum nested within a clade comprising solitary peritrichs and was distantly related to the type species of Carchesium, C. polypinum. These findings imply for the first time that Carchesium is non-monophyletic.

Antibacterial effect and mechanism of anthocyanin from Lycium ruthenicum Murr.

The inhibitory effects of the anthocyanin obtained from Lycium ruthenicum Murr were tested against several food-borne pathogens were evaluated, such as Staphylococcus aureus (S. aureus), Escherichia coli (E. coli), Aspergillus niger and Penicillium sp. In general, anthocyanin had different antibacterial effect on different bacteria, and the best antibacterial effect on S. aureus, with minimal inhibitory concentration (MIC) of 3.125 mg/mL. Anthocyanin increased the surface hydrophobicity of S. aureus, discharged the intracellular K+, and reduced the total soluble protein, affecting protein synthesis. Fluorescent inverted microscope and flow cytometry (FCM) found a significant increase in fluorescence intensity and lethality relative to the control group, and the dead P3 region to 77.21%. The above suggested a correlation between the antibacterial mechanism of anthocyanin and cell membrane permeability integrity. Biofilm formation was evaluated by the crystal violet assay (CV), silver staining method and methyl thiazolyl tetrazolium (MTT). Scanning electron microscopy (SEM) showed that anthocyanins could change the structure of biofilm.

Morphology and SSU rRNA gene-based phylogeny of three peniculid ciliates (Ciliophora, Oligohymenophorea) from China, including a new Frontonia species

In the present study, a new freshwater peniculid species, Frontonia apoelegans sp. nov., and two other peniculid species, Frontonia atra (Ehrenberg, 1833) Bütschli, 1889 and Stokesia vernalis Wenrich, 1929, were isolated from Lake Weishan wetland, northern China. Their morphology and infraciliature are described based on live observations and silver staining methods. The SSU rRNA gene sequences are also provided. Frontonia apoelegans sp. nov. is recognized by the following combination of characteristics: two contractile vacuoles located right-dorsally, without collecting canals; peniculi 1 and 2 four-rowed, peniculus 3 three-rowed with leftmost row containing only one kinetosome; 62–76 somatic kineties; three ophryokineties; and four or five postoral kineties. We also provide improved diagnoses for Frontonia atra and Stokesia vernalis based on current and previous reports. Comparisons with sequences of morphologically similar species clearly support the validity of the new species. Phylogenetic analyses based on SSU rRNA gene sequence data reveal that Frontonia species with two contractile vacuoles cluster in a single clade, indicating these species may have a common origin. The family Frontoniidae is non-monophyletic whereas the family Stokesiidae remains monophyletic according to our analyses.

Evaluation of Histopathological Effects of Smoked Marijuana on Albino Rats and Its Oxidative Stress Indices

Smoking marijuana (cannabis Savita), a psychoactive substance may result in side effect on living cells of the system and oxidative stress may meditate some of these effects. This study aims to evaluate the acute and chronic histopathological effect on wistar albino rat as well as oxidative stress indices and some of the haematological parameters evaluation. This study was conducted at the animal house of Ebonyi State University, Presco campus Abakaliki. The experimental animals were share into control group, acute and chronic administered group which was housed in the temperature and humidity controlled facility of 22°C+1, standard laboratory mice chow diet with tap water freely available at ad libitum to the animals. The weight of the male wistar albino rat were taken twice daily which range from 150g to 200g after which was exposed to smoke of burnt wrapped 2mg of marijuana morning and evening for 21 days for experimental animals in group B and 42 days for group C animals respectively. The acute histopathological effect and that of chronic of the lungs of smoked marijuana wistar albino rat displayed histomorphological changes with Haematoxylin and Eosin, methanamine silver staining and phosphotungstic acid staining method when compared with the control lung sections that shows normal histomorphological pattern with the present of alveolar pneumocytes and alveolar space. Evaluation of the lungs of smoked marijuana rat shows increased MDA and decreased level of CAT, SOD, GSH and GPx but examining some of the haematological parameters show elevated Twbc and platelet while packed cell volume decreased the level as compared with control animals. I hereby concluded that marijuana smoked is a potent source of cellular oxidative stress that contributes significantly to cellular injury, dysfunction, and pulmonary edema as well as histomorphological changes from the lungs of marijuana smokers and disorder in some of the haematological parameters.

Improvement of the silver staining method for bacterial flagella

Flagella staining is a common method used in microbial research to identify and mark morphological features of bacteria. We improved the Blendon staining method by adding two steps to the usual procedure, viz. “preparation of a pre-atomized microscope slide” and “stretching flagella in situ”. The staining effects were then comparatively studied for this new, improved method on Bacillus subtilis, under four different culture conditions: 1) liquid culture medium, 2) aqueous solution at the bottom of slant medium, 3) solid culture medium adding water for stretching after culture, and 4) semi-solid culture medium adding water for stretching after culture. The results revealed that after the addition of these two steps to the usual procedure, the order of the staining effects for the four culture conditions from best to worst was as follows: semi-solid culture medium > solid culture medium > aqueous solution at the bottom of slant medium > liquid culture medium. Hence, the semi-solid culture medium brought about the best staining effect, with flagella stretching freely and not entangled with each other, while the liquid culture medium had the worst staining, owing to the serious background interference. This improved method is simple, low cost, and worthy of promotion.

Taxonomy and SSU rRNA gene-based phylogeny of two new Euplotes species from China: E. chongmingensis n. sp. and E. paramieti n. sp. (Protista, Ciliophora)

BackgroundThe genus Euplotes Ehrenberg, 1830, one of the most complicated and confused taxa, contains about 160 nominal species. It was once proposed to be divided into four genera, two of which were proved to be non-monophyletic. At least 19 new species have been discovered in the past decade, implying that there is a large undiscovered diversity of this genus.ResultsThe morphology of two new freshwater euplotid ciliates, Euplotes chongmingensis n. sp. and E. paramieti n. sp., isolated from Shanghai, China, were investigated using live observations, protargol staining, and Chatton-Lwoff silver staining method. Euplotes chongmingensis is characterized by its small size (40–50 × 25–35 μm), about 24 adoral membranelles, 10 frontoventral cirri, two marginal and two caudal cirri, eight dorsolateral kineties with 11–16 dikinetids in the mid-dorsolateral kinety and a double type of silverline system. Euplotes paramieti n. sp. is 180–220 × 110–155 μm in vivo and strongly resembles E. amieti but having a difference of 57 bp in their SSU rRNA gene sequences. Phylogenetic analyses based on SSU rRNA gene sequence data were used to determine the systematic positions of these new taxa.ConclusionsThe description of two new freshwater taxa and their SSU rRNA gene sequences improve knowledge of biodiversity and enrich the database of euplotids. Furthermore, it offers a reliable reference for environmental monitoring and resource investigations.

Microscopic Imaging and Labeling Dataset for the Detection of Pneumocystis jirovecii Using Methenamine Silver Staining Method

Pneumocystis jirovecii pneumonia is one of the diseases that most affects immunocompromised patients today, and under certain circumstances, it can be fatal. On the other hand, more and more automatic tools based on artificial intelligence are required every day to help diagnose diseases and thus optimize the resources of the healthcare system. It is therefore important to develop techniques and mechanisms that enable early diagnosis. One of the most widely used techniques in diagnostic laboratories for the detection of its etiological agent, Pneumocystis jirovecii, is optical microscopy. Therefore, an image dataset of 29 different patients is presented in this work, which can be used to detect whether a patient is positive or negative for this fungi. These images were taken in at least four random positions on the specimen holder. The dataset consists of a total of 137 RGB images. Likewise, it contains realistic, annotated, and high-quality microscope images. In addition, we provide image segmentation and labeling that can also be used in numerous studies based on artificial intelligence implementation. The labeling was also validated by an expert, allowing it to be used as a reference in the training of automatic algorithms with supervised learning methods and thus to develop diagnostic assistance systems. Therefore, the dataset will open new opportunities for researchers working in image segmentation, detection, and classification problems related to Pneumocystis jirovecii pneumonia diagnosis.

Re-description and molecular phylogeny of the free-swimming peritrichs Hastatella radians Erlanger, 1890 and H. aesculacantha Jarocki & Jakubowska, 1927 (Ciliophora, Peritrichia) from China

The present work investigated Hastatella radians Erlanger, 1890 (type species), and H. aesculacantha Jarocki & Jakubowska, 1927, isolated from Chongming Island, Shanghai, China, based on light microscopy observations of live individuals, silver staining and molecular phylogeny methods. Hastatella radians is characterized by its size (about 30–75 μm long, 25–70 μm wide), two girdles of ectoplasmic processes, infundibular polykinety 3 (P3) composed of two rows of unequal length and terminating beyond adstomal end of P2, 8–22 transverse silverlines between peristome and trochal band, and 5–9 between trochal band and scopula. The infraciliature of H. aesculacantha is described for the first time, and we improved its diagnosis: cell size about 25–52 × 20–41 μm in vivo; four girdles of ectoplasmic processes; P3 two-rowed, outer row much shorter than inner one; 5–7 silverlines between peristome and trochal band and four or five between trochal band and scopula. Phylogenetic analyses based on SSU rDNA sequences revealed that Hastatella is monophyletic and has a close relationship to Astylozoon enriquesi.

The History of Neuroscience 5: Discovery of the Neuron and Its Connections

Thanks to the invention of the microscope equipped with an achromatic lens, research on the histology of the nervous system flourished during the 19th century. Jan Evangelista Purkinje, together with Gabriel Valentin, first depicted individual nerve cells. Robert Remak, one of the most remarkable microscopists of the 19th century, made the pivotal discovery that the cell body (globule) and fibers arising from the globule formed a single unit. In addition to improvements in microscopes, a new method of visualizing nervous tissue was developed—namely, the silver staining method introduced by Camillo Golgi. Golgi could identify the types and delicate branches of axons. However, he believed that axons always directly merged with other axons, which was the basis of the reticular or nerve net theory. Santiago Ramón y Cajal, the founder of modern neuroscience, finally established the cornerstone of the neuron doctrine, according to which the nerve cell is the basic unit of the nervous system and information flows from unit to unit without direct fusion. This structure is called the synapse. Key words: Achromatic lens; Histology; Silver staining; Neuron doctrine; Synapses.

Molecular Data Reveal a Cryptic Diversity in the Genus Urotricha (Alveolata, Ciliophora, Prostomatida), a Key Player in Freshwater Lakes, With Remarks on Morphology, Food Preferences, and Distribution.

Species of the ciliate genus Urotricha are key players in freshwater plankton communities. In the pelagial of lakes, about 20 urotrich species occur throughout an annual cycle, some of which play a pivotal role in aquatic food webs. For example, during the phytoplankton spring bloom, they consume a remarkable proportion of the algal production. In ecological studies, urotrich ciliates are usually merely identified to genus rank and grouped into size classes. This is unsatisfying considering the distinct autecological properties of individual species and their specific spatial and temporal distribution patterns. As a basis for future research, we characterized in detail four common urotrich morphotypes, i.e., specimens identified as U. furcata and tentatively as U. agilis, U. pseudofurcata, and U. castalia, using state-of-the-art methods. We used an integrative polyphasic approach, in which morphological studies (in vivo observation, silver staining methods, scanning electron microscopy) were linked with a molecular approach exploiting four different gene fragments as taxonomic DNA barcodes with different resolution potential (SSU rDNA, ITS-1, ITS-2, hypervariable V4 and V9 regions of the SSU rDNA). We shed light on the diversity of urotrich ciliates as well as on their global distribution patterns, and annual cycles. Additionally, we coupled individual species occurrences and environmental parameters, and subsequently modeled the distribution and occurrence, using logistic regressions. Furthermore, for one strain putatively identified as U. castalia, we ascertained the optimal cultivation media and food preferences. Thereby, our comprehensive view on these important freshwater ciliates that frequently occur in environmental high throughput sequencing datasets worldwide will allow future studies to better exploit protistan plankton data from lakes.

Allergenic Proteins of Tilia Cordata

Pollen forms large amount of aeroallergens in the atmosphere that spread through winds and insects. Allergenic pollen grains are water-soluble glycoprotein or protein bioparticles and its weight is approximately 5-80 kDa. There are many factors that cause allergies and considering the recently changing climatic conditions and their ability to spread over a wide geographical areas, pollens are among the major factors causing allergies. In this study, it was aimed to detect potential allergen pollen proteins of Tilia cordata which is a potent allergen. The pollen samples belonging to T. cordata were dried and then they were separated with sieve. Acetone washing and dialysis processes were applied to purificate pollen. Afterwards, the amount of protein in the pollens of T. cordata was determined by the bicinchoninic acid (BCA) method. In order to detect allergen proteins, gel runing was performed by SDS-PAGE. Silver staining method was applied to make visible the bands of T. cordata pollen proteins obtained from the gel. Five different allergenic pollen proteins, weights 10, 23, 40, 50 and 80 kDa, were detected as a result of SDS-PAGE. This is the second study in the literature related with the identification of T. cordata allergenic proteins and we showed two different protein bands compared with the former study result. Besides, our study is original in this perspective. We think that this study contributes to the literature on allergenic proteins of T. cordata and should be supported by future studies.

Allergenic Proteins of Tilia Cordata

Pollen forms large amount of aeroallergens in the atmosphere that spread through winds and insects. Allergenic pollen grains are water-soluble glycoprotein or protein bioparticles and its weight is approximately 5-80 kDa. There are many factors that cause allergies and considering the recently changing climatic conditions and their ability to spread over a wide geographical areas, pollens are among the major factors causing allergies. In this study, it was aimed to detect potential allergen pollen proteins of Tilia cordata which is a potent allergen. The pollen samples belonging to T. cordata were dried and then they were separated with sieve. Acetone washing and dialysis processes were applied to purificate pollen. Afterwards, the amount of protein in the pollens of T. cordata was determined by the bicinchoninic acid (BCA) method. In order to detect allergen proteins, gel runing was performed by SDS-PAGE. Silver staining method was applied to make visible the bands of T. cordata pollen proteins obtained from the gel. Five different allergenic pollen proteins, weights 10, 23, 40, 50 and 80 kDa, were detected as a result of SDS-PAGE. This is the second study in the literature related with the identification of T. cordata allergenic proteins and we showed two different protein bands compared with the former study result. Besides, our study is original in this perspective. We think that this study contributes to the literature on allergenic proteins of T. cordata and should be supported by future studies.

POLYMORPHISM OF TAGLGAP MICROSATELITE LOCUS AND ITS CONNECTION WITH ALLELELIC VARIETIES OF GLIADINS OF BREAD WHEAT

Introduction. Gliadins are monomeric and highly polymorphic storage proteins of wheat endosperm, which together with glutenins form a gluten complex that determines the breadmaking properties of wheat. Allelic variants of gliadins are an important feature in the selection of material for breeding, but their determination by electrophoresis in acid PAGE is quite difficult. Aim. The aim of this study was to investigate the polymorphism of the Taglgap microsatellite locus and to analyze its correspondence to the polymorphism of allelic variants of gliadins that have been revealed by acid PAGE electrophoresis. Methods. 140 cultivars and lines of bread wheat of Ukrainian and foreign selection were analyzed. Electrophoresis of storage proteins was performed in an acid PAGE according to the method of F. O. Poperellia (1989), allelic variants were designated according to the international nomenclature (Metakovsky et al., 2018). DNA was isolated by CTAB method and PCR was performed with primers to the Taglgap microsatellite (Devos et al., 1995). PCR products were fractionated in 7% PAGE and stained with silver staining method. Nucleotide sequences were searched by BLAST and aligned by MAFT methods. The main results. 19 allelic variants of gliadins and 11 alleles of the Taglgap locus were identified. In the collection of Ukrainian varieties there were Gli-B1b, Gli-B1c, Gli-B1d, Gli-B1e, Gli-B1f, Gli-B1g, Gli-B1h, Gli-B1l and Gli-B1o allelic variants and alleles of Taglgap 216 bp, 237 bp, 246 bp, 248 bp, 252 bp, 267 bp, 270 bp and null. In the foreign collection of varieties − Gli-B1a, Gli-B1b, Gli-B1c, Gli-B1d, Gli-B1e, Gli-B1f, Gli-B1g, Gli-B1h, Gli-B1i, Gli-B1j, Gli-B1k, Gli -B1l, Gli-B1m, Gli-B1n, Gli-B1o, Gli-B1p, Gli-B1q, Gli-B1r, Gli-B1s and 213 bp, 216 bp, 237 bp, 246 bp, 248 bp, 250 bp, 252 bp, 270 bp, 285 bp and null. Nucleotide sequence analysis in the NCBI database showed the presence of a number of other alleles of the Taglgap microsatellite not only in bread wheat but also in some species of the Triticum L. and Aegilops L. genus. Conclusions. The detected polymorphism correlates with the polymorphism of allelic variants of gliadins of Gli-B1 locus and makes it possible to identify Gli-B1a, Gli-B1d, Gli-B1h and Gli-B1l allelic variants, and for Ukrainian varieties with high probability also Gli-B1b allelic variant. However, this marker does not allow identifying Gli-B1c, which is important for selection.

Morphological and morphometric changes in the Purkinje cells of patients with essential tremor.

Essential tremor (ET) is a progressive neurological syndrome characterised by involuntary tremors of the hands or arms, head, jaw and voice. The pathophysiology of ET is not clearly understood yet. However, previous studies have reported several changes in the brain of patients with ET. One of the brain areas extensively investigated is the cerebellum. In the present study, a morphometric analysis of Purkinje cells in patients with ET and ET-plus was performed, and subsequently compared with normal controls using the Golgi silver staining method and 3D neuronal reconstruction. Substantial morphological changes were uncovered in the Purkinje cells of patients with ET compared with normal controls, including a decreased dendritic length and field density, an overall loss of terminal branches and a decreased density of dendritic spines.

Tetrahymena pyriformis infection in the tiger barb, Puntius tetrazona: Parasite characterization and pathology of infected fish

Tetrahymena pyriformis is a histophagus protozoan ciliate responsible for tetrahymenosis in various aquatic organisms including the tiger barb Puntius tetrazona. The morphology, infraciliature, SSU rRNA and cox 1 gene sequences of T. pyriformis isolated from P. tetrazona from a fish market in Harbin was investigated using living observation and silver staining methods. In addition, an SSU-rDNA-targeted oligonucleotide probe labelled with a fluorochrome was designed for rapid identification of T. pyriformis. Diseased fish secreted copious amounts of mucus on the skin and had dark-red spots in the eyes and at the base of tail fins. Gills of all diseased fish were shriveled and pale. T. pyriformis could be directly found when examined histologically by hematoxylin-eosin staining. The results showed that some T. pyriformis cells were present on the surface of the gill filaments. There was also abnormal bunting of the abdomen, and the gill filaments and muscle tissue were deformed, shrunken, and cracked. Successful infection of P. tetrazona was achieved with T. pyriformis by scratch inoculation with a range of infectious doses. The results indicate that T. pyriformis invades P. tetrazona through lesions on the body surface, and a large infectious dose could accelerate the death of the infected fish.

Taxonomy and molecular phylogeny of a new freshwater ciliate Frontonia apoacuminata sp. nov. (Protista, Ciliophora, Oligohymenophorea) from Qingdao, PR China.

The morphology and ciliature of a new freshwater ciliate, Frontonia apoacuminata sp. nov., isolated from an artificial pond in Qingdao, PR China, were investigated using live observation and silver staining methods. The main features separating F. apoacuminata sp. nov. from its congeners are as follows: a broad elliptical body that is slightly pointed at the posterior end, four ophryokineties, one or two spherical micronuclei of a 'compact' type, a dorsally positioned contractile vacuole, and peniculi 1-3 each with five kinetosome rows though the left-most two rows in peniculus 3 are extremely shortened (with only two or three kinetosomes each). Additionally, an improved diagnosis of F. acuminata is provided. Phylogenetic analyses based on the small subunit ribosomal RNA (SSU rRNA) gene show that F. apoacuminata sp. nov. clusters with F. atra, F. minuta, F. acuminata and F. terricola. These five species group with Disematostoma, Marituja and Stokesia rather than with other Frontonia species, causing polyphyly of the genus Frontonia.

Analyses of the Updated "Animal rDNA Loci Database" with an Emphasis on Its New Features.

We report on a major update to the animal rDNA loci database, which now contains cytogenetic information for 45S and 5S rDNA loci in more than 2600 and 1000 species, respectively. The data analyses show the following: (i) A high variability in 5S and 45S loci numbers, with both showing 50-fold or higher variability. However, karyotypes with an extremely high number of loci were rare, and medians generally converged to two 5S sites and two 45S rDNA sites per diploid genome. No relationship was observed between the number of 5S and 45S loci. (ii) The position of 45S rDNA on sex chromosomes was relatively frequent in some groups, particularly in arthropods (14% of karyotypes). Furthermore, 45S rDNA was almost exclusively located in microchromosomes when these were present (in birds and reptiles). (iii) The proportion of active NORs (positively stained with silver staining methods) progressively decreased with an increasing number of 45S rDNA loci, and karyotypes with more than 12 loci showed, on average, less than 40% of active loci. In conclusion, the updated version of the database provides some new insights into the organization of rRNA genes in chromosomes. We expect that its updated content will be useful for taxonomists, comparative cytogeneticists, and evolutionary biologists.